Diabetic model

ABSTRACT

The invention relates to an animal model for diabetes and a method for obtaining said animal model. The invention also relates to the uses of the animal model for screening for or testing compounds for treating or preventing diabetes symptoms. The invention further relates to an assay for determining an individual&#39;s susceptibility to developing diabetes. The invention also relates to nucleic acid molecules isolated from Ljungan virus and to polypeptides encoded by any portion of said nucleic acid molecule.

[0001] This application claims priority under 35 U.S.C. § 119 fromUnited Kingdom application number 0120437.9, filed Aug. 22, 2001,incorporated herein in its entirety by reference.

TECHNICAL FIELD OF INVENTION

[0002] The present invention relates to a method for obtaining an animalmodel for diabetes. The present invention also relates to the uses ofthe animal model for screening for or testing compounds which affectdiabetic symptoms. The present invention also relates to an assay fordetermining an individual's susceptibility to developing diabetes.

BACKGROUND OF THE INVENTION

[0003] Diabetes is a disease in which the body does not produce or useinsulin correctly. Insulin is a hormone that is required to convertsugar, starches and other food into energy needed for daily life.Insulin is produced by the beta cells in the islets of Langerhans in thepancreas. Partial or total loss of these cells will result in partial ortotal loss of insulin production.

[0004] There are two major types of diabetes.

[0005] Type 1 diabetes is an autoimmune disease in which the bodyactually fails to produce any insulin. Type 1 disease most often occursin children and young adults but can develop at any age. Type 1 diabetesis characterized by total loss of beta cells so that the patientrequires insulin by injection. Type 1 diabetes accounts for 10-15% ofall diabetes. Type 1 diabetes is strongly associated withauto-antibodies and this association has become part of thedefinition/classification of type 1 diabetes. Type 1 diabetes isdiscussed in greater detail below.

[0006] Type 2 diabetes is a metabolic disorder resulting from the body'sinability to make enough, or properly use, insulin. It is the mostcommon form of the disease. Type 2 diabetes accounts for 85-90% ofdiabetes.

[0007] The definitions of type 1 and 2 diabetes, however, are changingslowly. Auto-antibodies are found in type 2 diabetes patients and type 2diabetes is found in increasing numbers in children. As a result, thetraditional view of type 1 and 2 diabetes as two different diseases bothresulting in increased blood glucose levels is shifting to the view thatthere is a large grey zone with patients in between the two extremes.This view is important when evaluating the usefulness of differentanimal models.

[0008] Both genetic and environmental factors are believed to beinvolved in the development of type 1 (insulin dependent) diabetes (forreviews see Leslie et al., Diabetologia, 42, 3-14, 1999; and Schranz etal., Diab. Metab. Rev., 14, 3-29, 1998). The HLA Class II region is thestrongest genetic component, but other genes and loci have beenimplicated as contributing to a genetic predisposition to the disease(reviewed in Schranz et al., 1998 (supra)). Monozygotic twin studiesshow only 20-30% concordance of type 1 diabetes indicating a significantcontribution of environmental factors (Kyvik et al., BMJ, 311, 913-7,1995). The role of environmental factors is also supported by the factthat more than 85% of new onset patients do not have a first degreerelative with the disease (Dahlquist et al., Diabetologia, 32, 2-6,1989).

[0009] Worldwide, there is a large variation in the incidence of type 1diabetes, ranging from more than 40 patients per 100,000 in Finland to1-2 cases per 100,000 in Japan (Onkamo et al., Diabetologia, 42,1395-403, 1999). Seasonal variation in incidence rate, together withserological studies, have suggested viral infections as a majorenvironmental risk factor for type 1 diabetes (for reviews see Jun etal., Diabetologia, 44, 271-285, 2001; Rayfield et al., Diab./Metab.Rev., 3, 925-57, 1987; and Vaarala et al., Diabetes Nutr. Metab., 12.,75-85, 1999). Congenital rubella virus infection (Menser et al., Lancet,i, 57-60, 1978) or different members in the enterovirus genus are mostoften implicated as an etiologic agents in diabetes development (Yoon,Do Viruses Play a Role in the Development of Insulin-dependentDiabetes?, 1991; Vaarala et al., 1999, (supra)). Signs of enterovirusinfection during pregnancy (Dahlquist et al., Diabetologia, 32, 2-6,1989; and Hyoty et al, Diabetes, 44, 652-657, 1995) and in some infantswho developed islet cell autoantibodies and later type 1 diabetes(Lonnrot et al., Diabetes, 49, 1314-8, 2000) further supports thishypothesis. Both Coxsackie B and rota virus contain peptide sequencesalso found in the islet autoantigens glutamate decarboxylase (GAD65)(Kaufman et al., J. Clin. Invest., 89, 283, 292, 1992), thetyrosine-phosphatase like protein IA-2 (Honeyman et al., Diabetes, 49,1319-1324, 2000) or proinsulin (Rudy et al., Mol. Med., 1, 625-33, 1995)suggesting that T lymphocytes recognizing viral antigens may potentiallycontribute to islet autoimmunity by cross-reactivity or molecularmimicry. Indeed, cross-reactive GAD65 and rubella virus peptides wererecognized by T cells in type 1 diabetes patients (Ou et al.,Diabetologia, 43, 750-62, 2000). Since T cell tests that predict type 1diabetes are not yet available, standardized tests for GAD65, IA-2 orinsulin autoantibodies are useful markers to predict type 1 diabetes(for a review see Gottleib et al., Arum. Rev. Med., 49, 391-405, 1998).Rota virus seroconversion was reported to be associated with increasesin autoantibodies to GAD65, IA-2, and insulin suggesting that this virusinfection may trigger or exacerbate islet autoimmunity in geneticallysusceptible children (Honeyman et al., 2000 (supra)). Coxsackievirus-induced diabetes in mice was also associated with the developmentof GAD antibodies (Gerling et al., Autoimmunity, 6 49-56,1991). It isstill controversial, however, whether viruses cause beta celldestruction directly by a cytolytic infection in the islets orindirectly by initiating autoimmunity (Vreugdenhil et al., Clin. Infect.Dis., 31, 1025-31, 2000; and Kukreja et al., Cell Mol. Life Sci., 57,534-41, 2000).

[0010] Rodents are well-known reservoirs and vectors for viruses causingdisease in humans. Puumala virus causing Nephropathia Epidemica(Myhrman, Nordisk Medicinsk Tidskrift, 7, 739-794, 1934; and Niklassonet al., Lancet, 1, 1012-3, 1984) is one example of an important humanpathogen carried by bank voles. It has been demonstrated that theincidence rate of human Nephropathia Epidemica correlates with the volepopulation density during the previous year (Niklasson et al., Am. J.Trop. Med. Hyg., 53, 134-40, 1995). More recently, statistical evidencesuggests that type 1 diabetes in humans also tracks the 3- to 4-yearpopulation density cycles of the bank vole with a similar time lag(Niklasson et al., Emerg. Infect. Dis., 4, 187-93, 1998). It also wasobserved that a high frequency of bank voles trapped in the wild andkept in the laboratory for studies of stereotypic behavior (Schoeneckeret al., Appl. Anim. Behav. Sci., 68. 349-357, 2000) develop symptoms oftype 1 diabetes, i.e., polydipsia and glucosuria, at a high frequency.

[0011] Currently there are two main animal models of diabetes: the NOD(non obese diabetic) mouse and the BB (bio breeding) rat. Both modelsinvolve animals with insulin dependent diabetes. Both of the currentmodels, however, fail to display important symptoms of human diabetes.The NOD mouse, for example, shows gender preferences that are oppositeto the human disease (i.e., more females than males develop thedisease), develops mild diabetes, requires a long time before developingketoacidosis, and fails to develop autoantibodies to GAD65, 1A-2 orinsulin. The disease is genetically controlled in the NOD mouse and thecleaner the animal, the higher the frequency of diabetes.

[0012] The BB rat is also no ideal. The animals have lymphopeniacontrolled by an autosomal mutation on chromosome 4 and the developmentof autoantibodies in inbred and specific pathogen free BB rats appearsnegligible. None of these BB rats develop diabetes in association withan infectious agent.

[0013] Thus, there is a need to develop an improved method for obtainingan animal model which displays the features of diabetes for bothresearch and therapeutic purposes.

SUMMARY OF THE INVENTION

[0014] The invention provides an animal model for human diabetes andmethods for producing it. The invention also provides methods forscreening for or testing compounds which affect diabetic symptomscomprising use of the animal model. The invention further provides anassay for determining an individual's susceptibility to developingdiabetes.

BRIEF DESCRIPTION OF DRAWINGS

[0015]FIG. 1 shows the histology of the pancreas in bank voles withoutdiabetes (panels a and b as well as e and f) and with diabetes (panels cand d as well as g and h). The histology of non-diabetic bank volesdemonstrates well-defined islets of Langerhans surrounded by aconspicuous and delicate capsule. Diabetic bank voles have dramaticislet cytopathology characterized by distinct vacuoles or fattyinfiltration of the pancreatic islets. Hematoxylin and Eosin stainingare shown in panels a-d while immunostaining for insulin and glucagonare shown in panels e-h. The immunostained sections demonstrate that thecytopathology affected only insulin positive cells, which were lostresulting in a redistribution of glucagon immunoreactive cells. Sizebars are indicated in each panel.

[0016]FIG. 2 shows the histology of the pancreas of a diabetic bank voledemonstrating islet infiltration of mononuclear cells followingHematoxylin and Eosin staining.

[0017]FIG. 3 shows the histology of the pancreas in bank voles without(panels a and b) and with diabetes (panels c and d) followingimmunostaining with the mouse 87-012 or 145L antiserum against Ljunganvirus. The binding of the mouse antiserum was revealed with red vectorstaining. The sections were double stained with glucagon antiserumrevealed with alkaline phosphatase and tetrazolium blue. Thenon-diabetic voles did not show binding of mouse anti-Ljungan virusantibodies while the immunostaining against glucagon stained cells inthe periphery of the islets (panel a). The islets in diabetic bank volesshowed varying degree of vacuolization or fatty infiltration of thepancreatic islets (panels b, c and d). The edges of these lesions arestained indicating the presence of Ljungan virus antigen. The glucagonimmunostaining showed redistribution of cells that became morepronounced the greater the lesions.

[0018]FIG. 4 shows that the bank voles have autoantibodies against isletcell autoantigens and against Ljungan virus in vitro translatedantigens. Autoantibodies to GAD65 (panel a), IA-2 (panel b), insulin(panel c) as well as Ljungan virus in vitro translated antigens areshown as in-house relative Units on a log scale (wherein a 1/25 dilutionof standard serum is equal to 100 units/ml). Group A animals were caughtand bled in the wild and only 4% had diabetes. Group B bank voles werecaptive and 33% of the animals shown had diabetes. The levels of GAD65(p<0.0001), IA-2 (p<0.0001) and insulin (p<0.03) autoantibodies wereincreased in Group B compared to Group A bank voles. The autoantibodylevels of both GAD65 and IA-2 were higher in diabetic as compared tonon-diabetic Group B bank voles as indicated in the Figure. The data inpanel d demonstrate that antibodies to Ljungan virus in vitro translatedantigens were also increased in diabetic compared to non-diabetic bankvoles. Data for individual bank voles are shown.

[0019]FIG. 5 shows the sequence similarities and cross-reactivitybetween GAD65 autoantibodies and mouse or human anti-Ljungan virusantibodies. Sequence comparisons between the predicted amino acidsequence of Ljungan virus (serotype 87-012) and type 1 diabetesassociated autoantigens are shown in panel a. The data compares the87-012 Ljungan virus sequence and regions of potential molecular mimicryto GAD65, IA-2 and insulin. Areas of homology are boxed, with identicalamino acids indicated by a dot, similar amino acids are boxed, andnon-similar amino acids are plain type. Antibodies against Ljungan virusraised in mice (antiserum 87-012) showed cross-reactivity with humanGAD65 (panel b). Radiobinding analysis to the 87-012 antiserum showedconcentration-dependent binding of ³⁵S-labeled Ljungan virus in vitrotranslated antigen (x-x) and human (o-o) but not mouse (•-•) GAJD65. Thecompetition at half maximal binding of the 87-012 antiserum betweenbinding of ³⁵S-labelled human GAD65 and unlabelled Ljungan virus antigen(x-x), human GAD65 (o-o) or human proinsulin (▾-▾) (panel c)demonstrates displacement by unlabelled Ljungan virus in vitrotranslated antigens as well as by recombinant human GAD65. FIG. 5d.shows binding of different ³⁵S-labeled antigens including Ljungan virusantigen (x-x), human GAD65 (o-o) or mouse GAD65 (•-•) to the type 1diabetes human serum #591 (panel d). Human and mouse GAD65 bind equallywell and there are also significant levels of antibodies detecting theLjungan virus in vitro translated antigens. Competition at half maximalbinding to human serum 591 of ³⁵S -labelled Ljungan virus antigen andcold Ljungan virus antigen (x-x), human GAD65 (o-o) or human proinsulin(▾-▾) (panel e) showed displacement of cold Ljungan virus in vitrotranslated antigens but not of cold GAD65 or proinsulin. Allradioactivity values in cpm are mean values±SEM for 3-5 experiments TheSEM bars are within the size of the symbols unless indicated.

[0020]FIG. 6 shows the results of tests on sera from children with newonset type 1 diabetes indicating the presence of anti-Ljungan virusantibodies. Ljungan virus antibodies were determined in two independenttests by either indirect immunofluorescence of cells used to propagatethe virus or by the radioligand binding assay with Ljungan virus invitro translated antigens. The radioligand binding assay correlated tothe indirect immunofluorescence test (p<0.001 at 95 confidenceinterval).

[0021]FIG. 7 shows the nucleotide sequence of Ljungan virus 87-012.

[0022]FIG. 8 shows the nucleotide sequence of Ljungan virus 145SL.

[0023]FIG. 9 shows the nucleotide sequence of Ljungan virus 174F.

DETAILED DESCRIPTION OF THE INVENTION

[0024] According to a first embodiment of the present invention, thereis provided a method for obtaining an animal model for human diabetes,comprising obtaining a mammal that has been determined to be infectedwith Ljungan virus.

[0025] The mammal may be any mammal including rodents such as rats,mice, hamsters, guinea pigs, rabbits, bank voles and field voles; cattlesuch as cows; cats; dogs; and non-human primates. Preferably the mammalis a rodent, a cat or a dog, more preferably the mammal is a rodent,most preferably the mammal is a bank vole.

[0026] It has been found that bank voles having type 1 diabetes are allinfected with Ljungan virus and that the presence of Ljungan viruscauses or at least contributes to the development of type 1 diabetes.

[0027] The term “Ljungan virus” as used herein refers to any Ljunganpicornavirus as defined in International PCT patent application WO98/11133, the disclosure of which is incorporated herein by reference.Preferably the Ljungan virus is Ljungan virus 87-012, the nucleotidesequence of which is shown in FIG. 7; Ljungan virus 145SL, thenucleotide sequence of which is shown in FIG. 8; or Ljungan virus 174F,the nucleotide sequence of which is shown in FIG. 9.

[0028] The presence of Ljungan virus can be determined using anystandard procedure including, but not limited to, virus isolation,detection of Ljungan virus antigen by ELISA or immunohistochemistryusing antibody molecules having affinity for Ljungan virus or detectionof Ljungan virus specific RNA sequences using PCR or by a labelednucleic acid probe capable of specifically hybridizing to Ljungan virusnucleic acid. The presence of Ljungan virus also can be determined bydetecting for the presence of Ljungan virus antibodies using a suitabletest. Suitable techniques for determining the presence of Ljungan virusor anti-Ljungan virus antibodies are described in the examples below.

[0029] Use of the mammal obtained by the method according to the firstembodiment of present invention as a model for human diabetes has anumber of advantages over the prior art animal models of diabetesincluding:

[0030] a pathology that includes total destruction of the beta cellswithout affecting the surrounding pancreas tissue;

[0031] no or minor signs of inflammatory cells and only modestinsulitis; and

[0032] the presence of auto-antibodies used as markers for human type 1diabetes (antibodies to GAD 65, IA-2 and insulin) in most bank volesobtained by the method according to the first embodiment of the presentinvention.

[0033] The fact that the mammal obtained using the method according tothe first embodiment of the present invention has features that mimicthe human disease means that it closely represents the human disease andis therefore a particularly useful model of diabetes.

[0034] Preferably, the method according to the first embodiment of thepresent invention also comprises determining whether the mammal has highblood glucose levels that can be reduced by insulin and signs ofketoacidosis.

[0035] Preferably, the mammal is a bank vole. The bank vole may be anyspecies of bank vole. Preferably the bank vole is Clethrionomysglareolus. The mammal can be male or female. The bank vole may beobtained from the wild or may be the progeny of a bank vole obtainedfrom the wild. It is preferred that the bank vole obtained from the wildis obtained from Denmark, Sweden or Finland. Alternatively, the bankvole may be a laboratory bred bank vole.

[0036] The term “diabetes” as used herein means type 1 or type 2diabetes or diabetes having a combination of symptoms of both type 1 andtype 2 diabetes. The type of diabetes developed by the mammal willdepend on the type of mammal. For example bank voles infected withLjungan virus develop type 1 diabetes, whereas cats and dogs can developtype 1 or type 2 diabetes or diabetes having a combination of symptomsof both type 1 and type 2 diabetes. It is currently believed thatdiabetes in humans is not always type 1 or type 2 diabetes but thatdiabetes can fall somewhere between the two defined types wherein theindividual has some symptoms of both type 1 and type 2 diabetes. Theterm “diabetes” as used herein refers to diabetes characterized by highblood glucose levels that can be reduced by insulin and signs ofketoacidosis. The presence of auto-antibodies to at least one of GAD65,IA-2 and insulin is an additionally preferred characteristic of type 1diabetes according to the present invention. Additional features ofdiabetes such as hyperlipidemia, slowly progressive increase ofhyperglycemia and variable glucosuria as well as symptoms of hyperphagiaand obesity also may be present in addition to the characteristics oftype 1 diabetes defined above in accordance with the situation inhumans.

[0037] The term “high blood glucose levels” as used herein means bloodglucose levels that are at least 1.5 times as high, more preferably atleast 3 times as high and most preferably at least 5 times as high asthe mean level of blood glucose found in the corresponding non-diabeticmammals. Non-diabetic mammals are mammals that do not show any symptomsof diabetes such as increased glucosuria. It is particularly preferredthat a high blood glucose level is at least 150 mg/dl, more preferablyat least 200 mg/dl.

[0038] The term “reduced by insulin” as used herein means that the highblood glucose levels can be reduced by the addition of insulin.Preferably the blood glucose levels can be reduced by about 30%, morepreferably 60% and most preferably to approximately the level of anon-diabetic mammals by the addition of insulin. As those skilled in theart will appreciate, the reduction in blood glucose levels will varydepending on the amount of insulin given to the mammal.

[0039] Signs of ketoacidosis include nausea, vomiting, stomach pain,deep and rapid breathing, flushed face, dry skin and mouth, fruitybreath odor, rapid and weak pulse, low blood pressure. Ketoacidosis canbe determined by the measurement of keton bodies in the blood or inplasma or serum.

[0040] The method according to the first embodiment of the presentinvention preferably additionally comprises modulating the immune systemof the mammal to facilitate the development of diabetes. The modulationcan be by suppressing or enhancing the immune system. The immune systemof the mammal can be modulated by any method including administratingimmunosuppressing or immunostimulating agents, altering the diet of themammal or subjecting the mammal to stress. Preferably the immune systemof the mammal is modulated by subjecting the mammal to stress. Themammal can be subjected to any form of stress that affects the immunesystem of the mammal including keeping the mammal in a cage. Inembodiments in which the mammal is a bank vole, it preferably is kept ina cage for at least 2 months, more preferably at least 3 months.Preferably the mammal is kept isolated in its own cage.

[0041] The present invention also provides the use a mammal infectedwith Ljungan virus as a model of diabetes.

[0042] The mammal used as a model of diabetes is preferably obtained bythe method according to the first embodiment of the present invention.

[0043] The mammal can be used as a model of diabetes in order toinvestigate the development and etiology of diabetes. The mammal canalso be used to test candidate compounds for their effects on symptomsof diabetes. In particular, a candidate compound can be administered tothe mammal and the effects of the compound on symptoms of diabetes, suchas blood glucose levels, signs of ketoacidosis and glucosuria can bemeasured.

[0044] The mammal can also be used to screen for compounds having aneffect on the development of diabetes. Preferably the mammal is used toscreen for compounds that prevent the development of, or reduce thesymptoms of, diabetes.

[0045] The present invention also relates to the use of compoundsidentified in the above-described screening in the manufacture of acomposition for treating and/or preventing diabetes.

[0046] The present invention also relates to the use of cells obtainedfrom mammals obtained by the method according to the first embodiment ofthe present invention. The cells can be used in a variety of in vitroassays which are well known to those skilled in the art.

[0047] In a second embodiment of the present invention there is provideda method for producing diabetes in a mammal comprising infecting themammal with a Ljungan virus.

[0048] It has been found that mammals infected with Ljungan virusdevelop diabetes. The mammal infected with Ljungan virus can be used asa model of diabetes whether or not symptoms of diabetes can be detected.

[0049] The mammal can be infected using any standard technique,including, but not limited to, parenteral routes such as intravenousinjection and intraperitoneal injection. Methods for determining thenecessary viral dose leading to the development of diabetes can beeasily determined by those skilled in the art. In making such adetermination, a number of factors are considered including the speciesof mammal, the rate of viral replication, the route of infection, theage and sex of the mammal. Preferably about 1,000 infection units aregiven to the mammal.

[0050] The mammal infected with Ljungan virus may develop type 1 or type2 diabetes or diabetes having a combination of symptoms of both type 1and type 2 diabetes.

[0051] The method according to the second embodiment of the presentinvention preferably additionally comprises modulating the immune systemof the mammal as described above with respect to the first embodiment ofthe present invention. Preferably the immune system of the mammal iscompromised by subjecting the mammal to stress as described above withrespect to the first embodiment of the present invention subsequent toinfection with the infectious agent.

[0052] By compromising the immune system of the mammal, it has beenfound that the animals develop diabetes more quickly. Without beingbound to any one theory, it is believed that the Ljungan virus canreplicate at a faster rate leading to the development of diabetes in ashorter period of time in immune-compromised animals.

[0053] The present invention also provides the use of a mammal infectedwith a Ljungan virus as a model of diabetes. Preferably the mammal isobtained by the method according to the second embodiment of the presentinvention.

[0054] The mammal infected with a Ljungan virus can be used as a modelto investigate the development and etiology of diabetes. The mammal alsocan be used to test candidate compounds for their effects on diabetes.In particular, a candidate compound can be administered to a mammalinfected with a Ljungan virus and the effects of the compound onsymptoms of diabetes, such as blood glucose levels, signs ofketoacidosis, glucosuria, hyperlipidemia, a slowly progressive increasein hyperglycemia, symptoms of hyperphagia, obesity and insulinresistance, can be measured.

[0055] The mammal infected with a Ljungan virus can also be used toscreen for compounds having an effect on the development of diabetes.Preferably the mammal is used to screen for compounds which prevent thedevelopment of, or reduce the symptoms of, diabetes.

[0056] The present invention also provides an assay for determining anindividual's susceptibility to developing diabetes comprising analyzinga sample from the individual in order to determine if the individual isinfected with a Ljungan virus, wherein infection with a Ljungan virusindicates a greater susceptibility to developing diabetes.

[0057] It has been found that children with an increased level ofantibodies against Ljungan virus have type 1 diabetes (i.e.,serologically positive for Ljungan virus infection). We have determinedthat a population of children with diabetes has a much higher frequencyof being serologically positive for Ljungan virus than in a populationof healthy control children.

[0058] The presence of Ljungan virus can be determined using anystandard procedure including immunohistochemistry using antibodymolecules having affinity for Ljungan virus or by using a labelednucleic acid probe capable of specifically hybridizing to Ljungan virusnucleic acid. Alternatively the presence of Ljungan virus can bedetermined by detecting the presence of anti-Ljungan virus antibodiesusing a suitable test. Suitable techniques for determining the presenceof Ljungan virus or anti-Ljungan virus antibodies are described in theexamples below.

[0059] The present invention also provides a method of treating anindividual who has developed diabetes or is susceptible to developingdiabetes comprising administering an effective amount of a compoundwhich prevents or reduces Ljungan virus-induced diabetes.

[0060] Compounds which prevent or reduce the effects of Ljungan virusinclude antibody molecules having affinity for Ljungan virus or anyother anti-viral agents. Methods for producing suitable antibodymolecules are well know to those skilled in the art.

[0061] The present invention also provides a method of vaccinating anindividual against a Ljungan virus infection, thereby preventing, atleast in part, the individual developing diabetes.

[0062] Vaccines of the invention may comprise any antigenic portion ofthe Ljungan virus (e.g. a protein displayed on the surface of the virus)or by using an attenuated form of the Ljungan virus. Methods forproducing vaccines based on antigenic components or attenuated forms ofthe virus are well known to those skilled in the art and are describedin a variety of literature know to those skilled in the art (seeTextbook Field's Virology by David M. Knipe et al).

[0063] In another aspect, the invention includes nucleic acid moleculesisolated from Ljungan viruses or any portion thereof. In someembodiments, the nucleic acid is the one shown in any one of FIGS. 7, 8or 9 or any portion thereof.

[0064] The invention further comprises a nucleic acid molecule encodinga Ljungan virus polypeptide, or fragments thereof. In some embodiments,the nucleic acid is operably linked to one or more expression controlsequences. In some embodiments, the nucleic acid molecule or fragment isincorporated into a vector. In some embodiments, the vector is anexpression vector.

[0065] The invention also provides host cells comprising a nucleic acidor vector of the invention. The host cell can be prokaryotic oreukaryotic. The choice of host cells for expressing a Ljungan viruspolypeptide is well-known in the art.

[0066] The invention further provides methods for producing a Ljunganvirus polypeptide or fragment thereof comprising culturing host cells ofthe invention under conditions suitable for the expression of thepolypeptide and recovering the polypeptide.

[0067] The invention further comprises a vaccine comprising at least oneLjungan virus polypeptide or an immunogenic fragment thereof. In someembodiments, the vaccine comprises a plurality of Ljungan viruspolypeptides or immunogenic fragments thereof. The Ljungan viruspolypeptides can be from the same or different strains of Ljungan virus.Vaccines comprising polypeptides from different strains are useful toprevent or inhibit infection by a broader range of Ljungan virus inconditions caused by Ljungan virus infection, including diabetes.

[0068] In some embodiments, Ljungan virus polypeptides are a componentof a multivalent vaccine that comprises one or more components fromother pathogens, including human pathogens.

[0069] In some embodiments, a vaccine of the invention comprises anadjuvant. Methods for selecting an adjuvant for use in the vaccine arewell-known to those of skill in the art.

[0070] The invention further provides an antibody that specificallybinds Ljungan virus or a Ljungan virus polypeptide. In some embodiments,the antibody specifically binds one or more of the Ljungan viruspolypeptides shown in FIGS. 7-9.

EXAMPLES

[0071] In order that this invention may be better understood, thefollowing examples are set forth. These examples are for the purposes ofillustration only and are not to be construed as limiting the scope ofthe invention in any manner.

Materials and Methods

[0072] Wild caught bank voles (Group A). Group A bank voles represent101 animals from a single trapping session. These bank voles were testedat the trap for glucosuria and then euthanized. Heart-blood samples forblood glucose, ketosis, lipids and antibody analyses were takenimmediately after the voles were killed. Blood samples were eitherimmediately analyzed for blood glucose and ketones or centrifuged for 25minutes at 1,000×g and plasma stored at −30° C. Pancreas was dissectedand fixed in 4% paraformaldehyde followed by ethanol before beingembedded in paraffin.

[0073] Voles caught in the wild and kept in the laboratory (Group B). Intwo other trapping sessions, 163 voles were caught and transferred tothe laboratory as previously described (Schoenecker et al., Appl. Anim.Behav. Sci, 68, 339-347, 2000; Schoenecker et al., Appl. Anim. Behav.Sci., 68. 349-357, 2000). The animals were housed individually in smallbarren cages of transparent plastic (13.5×16.0×22.5 cm) under conditionsof minimum extraneous disturbance and with a twelve-hour light regime(8.00-20.00 h). The cages were supplied with a woodcutting bed, and food(standard rat chow) and water were available ad libitum. Cage cleaningand body weight measurements were performed once every week. A portionof grain mixture was given when the cages were cleaned. Diabetesdevelopment was followed by measurements of water intake, glucosuria,and blood glucose and ketonemia determined after bleeding from the retroorbital plexus. Polydipsic voles were characterized by >21 ml/day waterintake compared with non-polydipsic voles for which daily intake did notexceed 12 ml.

[0074] Histological analysis and immunocyto-chemistry. Standardhematoxylin and eosin staining was carried out on samples fixed in 4%paraformaldehyde, embedded in paraffin, cut into 5 micron sections, andaffixed to slides. Sections were deparaffinized, rehydrated, and stainedfor three minutes in Gill's hematoxylin and for one minute in Eosin Y.Stained sections were dehydrated and mounted.

[0075] In the immunohistochemistry tests, pancreas fixed in 4%paraformaldehyde and embedded in paraffin were cut into 5 micron thicksections, affixed to slides, deparaffinized and rehydrated. The sectionswere blocked for 30 min at RT in PBS containing 0.05% Tween 20 (Sigma,St. Louis, Mo.), 1% BSA (Sigma), 2% normal horse (in the case ofstaining for Ljungan virus antisera) or 2% normal goat (in the case ofinsulin or glucagon staining) serum (Vector Laboratories, Burlingame,Calif.), and 4 drops/ml Avidin solution (Avidin/Biotin blocking kit,Vector Laboratories). The primary antibody was diluted in PBS with 0.05%Tween 20, 1% BSA, 2% normal serum, and 4 drops/ml Biotin solution (fromAvidin/Biotin blocking kit, Vector Labs) to 1:100 (guinea piganti-insulin and rabbit anti-glucagon, Zymed Laboratories, S. SanFrancisco, Calif.) or 1:500 (mouse Ljungan virus antiserum). Slides wereexposed to the primary antibody solution for 60 minutes at roomtemperature or overnight at 4° C. Slides were then washed in PBS,incubated for 30 minutes at room temperature with a biotinylatedsecondary antibody (goat anti-rabbit IgG, goat anti-guinea pig IgG, orhorse anti-mouse IgG (Vector Labs) diluted 1:500 in PBS, and washedagain. The slides were next incubated for 30 minutes at room temperaturein alkaline phosphatase streptavidin conjugate (Vector Labs) at a 1:200dilution, washed in PBS, and reacted with the Vector Red or VectorBCIP/NBT alkaline phosphatase substrate kit. Finally, slides werecounter stained with methyl green, dehydrated, and mounted. All slideswere coded and scored independently by two readers.

[0076] Immunfluorescence assay for Ljungan virus antibodies. Sera fromchildren with type 1 diabetes and controls were tested for presence ofantibodies to Ljungan virus using an indirect immunofluorescence test(IFT). A previously described IFT protocol (Niklasson et al., J. Infect.Dis., 155, 369-76, 1987) was used to test antibody titers. Briefly, spotslides were prepared by incubating virus in Green Monkey Kidney cellsfor 8-10 days. At signs of discrete cytopathic effects (CPE), cells wereremoved from the flask with a rubber policeman and applied ontomicroscope slides, air dried, fixed in cold (4° C.) acetone and storedat −70° C. until used. The titer was determined after incubating theserum, diluted in PBS, on the slides at 37° C. for 1 h in a moistchamber and bound antibodies were detected by incubating FITC-conjugatedgoat anti-human IgG (Sigma, St Louis, Mo.) for 1 h at 37° C. Patient andcontrol sera was first tested at a 1:8 dilution using three Ljunganvirus isolates (87-012, 145SL, 174F). Any sera scoring positive for anyof the three isolates were titrated again using all three isolatesseparately. Patients and controls positive to one or several isolates ata titer of 32 or higher was considered positive.

[0077] Radioligand binding assays for GAD65 and IA-2 antibodies. GAD65and IA-2 antibodies were analyzed as described (Grubin et al.,Diabetologia, 37, 344-350, 1994; Hampe et al., J. Clin. Endocrinol.Metab., 85, 4671-9, 2000; Vandewalle et al., Diabetes Care, 20,1547-1552, 1997). GAD65 and IA-2 antibody levels were expressed in U/mlfor GAD65 and IA-2 antibodies using the WHO/JDF standard (Mire-Sluis etal., Diabetologia, 43, 1282-1292, 2000).

[0078] Insulin autoantibodies (IAA). IAA were measured using a methodfor small plasma/serum samples (Williams et al., Journal ofAutoimmunity, 10, 473-478, 1997). An in-house serum sample was used asthe standard to express the data in arbitrary U/ml. Recombinant humaninsulin (Novo Nordisk, Copenhagen, Denmark) was used to determine IAAspecificity as described (Williams et al., (supra), 1997).

[0079] Radioligand binding assay for Ljungan virus antibodies. We usedthe Ljungan virus cDNA (unpublished observation) in the coupled in vitrotranscription translation assay as described for GAD65 (Grubin et al.,(supra), 1994; Hampe et al., (supra), 2000). The Ljungan virus cDNA wastranslated into multiple components which were immunoprecipitated withLjungan virus mouse and guinea-pig antisera (data not shown) as well asfrom serum for both non-diabetic and diabetic bank voles and new onsettype 1 diabetic patients. The human 591 GAD65-positive serum (Mire-Sluiset al., (supra), 2000) showed high binding and was used as an in-housestandard to express antibody binding levels in arbitrary U/ml.

[0080] Competition experiments. Competition in binding betweenradioactive and cold antigens was carried out at half maximal binding ofeither the Ljungan virus 87-012 mouse antiserum or the 591 humanstandard serum found to be positive for antibodies against both Ljunganvirus in vitro translated antigens and GAD65 (Mire-Sluis et al.,(supra), 2000). Competition for binding of ³⁵S-labeled Ljungan virus invitro translated antigens was carried out with different concentrationsof unlabeled Ljungan virus in vitro translated antigens, recombinanthuman GAD65 (DiamydAB, Stockholm, Sweden) or human proinsulin (ElliLilly Company, Indianapolis, Ind.).

[0081] Type 1 diabetes patients and controls. A total of 53 childrenwith a median age of 10.1 years (range 2.3-16.4 years of age) werediagnosed with type 1 diabetes at the St Göran Hospital and AstidLindgren's Children's Hospital between 1992 and 1995. Within two days ofdiagnosis, blood samples were drawn for antibody analysis. Healthychildren (7 boys, median age 12.6 (7.8-16.8 years and 10 girls, medianage 13.5 (6.7-16.6 years ) were recruited from school classes in centralStockholm and children to personnel at the hospital. All children werepreviously healthy and without present medication. The Ethics Committeeat the Karolinska Institute, Stockholm, Sweden, approved the study.

[0082] Bioinformatics. To identify regions of high local homologybetween the virus polyprotein and known diabetes autoantigens, wecreated a local database of GAD65, IA-2 and insulin sequences and ranstand alone BLAST using software from the NCBI (Altschul et al., NucleicAcids Res., 25, 3389-402, 1997). Alignments were compiled manually toalign regions of similarity onto the Ljungan protein sequence usingCLUSTALW to determine similarity between non-homologous residues.

[0083] Statistics. The frequency of diabetes in the different groups wasanalyzed by Fischer exact test or Chi Square tests. Non-parametric testswere used to analyze differences in levels between groups. SpearmansRank Correlation was used to examine possible correlation betweendifferent parameters.

Example 1

[0084] Obtaining Bank Voles having Type 1 Diabetes

[0085] Development of Diabetes in Trapped Bank Voles

[0086] Bank voles were trapped from May to November in a forest habitaton the island of Zealand, Denmark. In different continuous trappingsessions of 30 days duration, 100 traditional live traps were set andinspected twice a day. Two groups of bank voles were analyzed fordiabetes and pancreas histology in addition to type 1 diabetesassociated autoantibodies against insulin (Williams et al., (supra),1997), GAD65 (Grubin et al, (supra), 1994), and IA-2 (Lan et al., DNAand Cell Biology, 13, 505-514, 1994) also known as ICA512 (Rabin et al.,J. Immunol, 152, 3183-3187,1994) as well as antibodies against Ljunganvirus (Niklasson et al., Virology, 255, 86-93, 1999). Group A bank volesrepresents 101 trapped bank voles that were euthanized in the forest forimmediate examination of blood glucose, glucosuria, body weight,pancreas histology and antibodies. Group B represents 67 bank voles thatwere trapped and kept in the laboratory for one month as previouslydescribed (Schoenecker et al., Appl. Anim. Behav. Sci., 68, 349-357,2000). An additional group of 54 animals were examined in Stockholm forinsulin sensitivity and pancreas histology before and after diabetesdevelopment.

[0087] The data in Table 1 shows the occurrence of diabetes in the twogroups of bank voles. In the Group A bank voles (n=101), four femaleanimals were found positive for glucosuria and blood glucose values of215, 302, 313 and 340 mg/dl, respectively, In the remaining bank voles,the mean blood glucose±S.D. was normally distributed at 101±28 mg/dl.The body weight of the trapped voles from group A ranged from 8.5-28.4g, the mean value±S.D. was 19±5 g. Occasional hyperglycemic andglucosuric bank voles may therefore be trapped in the wild. Whetherthese four animals had stress-induced hyperglycemia or overt diabetesremains to be established. These results in the Group A bank volesdiffer markedly from the 67 Group B bank voles that were trapped andkept in standard laboratory mouse cages for one month before they weretested for diabetes. We observed that 22/67 (33%) of these Group B bankvoles had a blood glucose above 200 mg/dl, the range being 211-540mg/dl. As many as 18/22 (82%) had ketones and were polydipsic. Genderdifferences are common in both humans (Harris, Diabetes in America, (ed.Harris, M. I.) (National Institutes of Health, Bethesda, 1995) and inanimal models of diabetes as well as in captured wild rodents thatdevelop non-insulin dependent diabetes when fed laboratory chow (for areview see (Shafrir et al., Diabetes Metab. Rev., 8, 179-208, 1992). Thebank voles we captured were also fed laboratory chow but they were notonly glucosuric and hyperglycemic but also positive for ketonuria,ketonemia and hyperlipidemia, all suggestive of type 1 diabetes (datanot shown). An insulin sensitivity test (Actrapid, Novo Nordisk,Copenhagen, Denmark) was also carried out in 16 randomly selectedStockholm bank voles to exclude diabetes due to insulin resistance. At60 minutes following insulin, four animals with blood glucose levelsabove 200 mg/dl experienced 30% decrease in blood glucose, four animalswith blood glucose at 120-200 mg/dl showed 60% decrease while eightanimals with blood glucose <120 mg/dl showed a 40% decrease in bloodglucose. These data indicate that bank voles with varying blood glucoselevels are insulin sensitive. We therefore next examined the pancreashistology in non-diabetic and diabetic bank voles to test if theclassification of type 1 diabetes was supported by a loss of beta cells.TABLE 1 The frequency of diabetes in wild caught bank voles and in bankvoles kept in the laboratory. Group of bank voles A. Analyzed B. Trappedand at trap captive N 101 67 M/F ratio 42/59 29/38 Blood glucose (mg/dl)Non-diabetic 101 ± 27  86 ± 24 Diabetic 293 ± 54 346 ± 88 Diabetes n (%)4 (4%) 22 (33%) M/F ratio 0/4 14/8 

[0088] Bank Voles Develop Type 1 Diabetes because of a Specific Loss ofBeta Cells.

[0089] The pancreas of all 101 Group A bank voles showed normal isletsas did those of non-diabetic Group B bank voles (FIG. 1). The fourhyperglycemic Group A bank voles had no appreciable islet lesions.Immunostaining with insulin and glucagon antibodies showed a normalislet cell distribution with beta cells located in the center surroundedby glucagon-positive cells (FIGS. 1a and 1 b). In dramatic contrast,Group B bank voles with diabetes had an almost complete loss ofcentrally located insulin-positive cells that were replaced by prominentvacuolization or fatty infiltration (FIGS. 1c and 1 d). Islets withinfiltrating mononuclear cells were occasionally observed (FIG. 2) butinsulitis was conspicuously absent in the majority of the bank voles.The beta cell destruction was unique to bank voles with diabetes andindicate that the animal should be classified as having type 1 diabetes.

[0090] In order to evaluate whether the Ljungan virus was associatedwith the islet beta cell lesion we next immunostained the pancreassections with high titer mouse antisera against Ljungan virus (Niklassonet al., (supra), 1999). We used antisera to two distinct Ljungan virusisolates, 87-012 and 145SL and, as controls, eight different antiseraprepared with the same procedure against Rift Valley Fever virus,Ockelbo virus, Langat virus and Sindbis virus. Both the 87-012 and the145SL Ljungan virus antisera at dilution of 1:4000 or higherimmunostained islets in diabetic but not in non-diabetic bank voles(FIG. 3) visualizing the presence of Ljungan virus antigen in affectedislets. None of the control sera showed immunostaining at a dilution of1:500 or higher. Furthermore, an analysis of Stockholm bank voleseuthanized with variable blood glucose levels after 2-3 months ofcaptivity revealed that the severity of beta cell loss was gradual (FIG.3 panel b, c and d). Also in these apparently early lesions, amononuclear cell infiltration was conspicuously absent. Without beingbound to any one theory, it is submitted that the beta cell-specificdestruction in association with immunoreactive virus antigen suggeststhat the Ljungan virus might have had a lytic effect on the beta cells,perhaps accelerated by the stress of bringing the bank voles intocaptivity. Although mononuclear cell infiltration was not a prominentfeature of the beta cell destruction it cannot be excluded that Ljunganvirus beta cell lysis results in autoantigen presentation that takesplace in lymph nodes draining the pancreas or by antigen presentingcells in or around the islets. We therefore next examined thepossibility that the development of diabetes was associated withautoantibodies to the islet autoantigens GAD65, IA-2 or insulin.Autoantibodies to these autoantigens predict type 1 diabetes in humans(reviewed in Schranz et al., (supra), 1998 and Leslie et al., (supra),1999) but not in the NOD mouse or BB rat models of type 1 diabetes (Bachet al., Endocrine Rev., 15, 516-542, 1994). The possible presence ofautoantibodies to these autoantigens would further support thehypothesis that the bank voles developed type 1 diabetes.

[0091] Bank Vole Diabetes is Associated with Autoantibodies to GAD65 andIA-2.

[0092] Standardized radioligand-binding assays that detectautoantibodies to GAD65 (Grubin et al., (supra), 1994; Hampe et al.,(supra), 2000), IA-2 (Kawasaki et al., Diabetes, 45, 1344-9, 1996;Vandewalle et al., (supra), 1997) and insulin (Williams et al., (supra),1997) were used to analyze serum samples from available animals of bothGroup A and Copenhagen group B bank voles (FIG. 4). Compared to Group Aanimals, GAD65 (P<0.001) but not IA-2 or insulin autoantibodies wereincreased non-diabetic Group B bank voles. More importantly, however,diabetic group B bank voles had higher GAD65 (P<0.001), IA-2 (P<0.001)and insulin (P<0.0346) autoantibody levels than the non-diabetic voles(FIG. 4). The increased levels of GAD65, IA-2 and insulin autoantibodiesfurther indicates that diabetes in these bank voles should be classifiedas type 1 diabetes.

[0093] Since Ljungan virus antigen was demonstrated in the islets ofdiabetic bank voles (FIG. 3) we next determined whether antibodies toLjungan virus antigens were associated to GAD65 and IA-2 autoantibodies.A radioligand binding assay, similar to the GAD65 and IA-2 autoantibodyassays (Grubin et al., (supra), 1994) was developed with ³⁵S-labelledvirus antigens generated by coupled in vitro transcription andtranslation using the T7 promoter of the Ljungan virus cDNA (unpublishedresults). Although the Group A bank vole sera showed a wide range ofantibody levels against Ljungan virus antigen (FIG. 4), the mean levelsof Ljungan virus antibodies in the non-diabetic Group B bank voles weresignificantly increased (P<0.001). In group B bank voles, the levels ofLjungan virus antibodies were higher in diabetic than non-diabeticanimals (P=0.0015). Since the diabetic Group B bank voles also showedincreased levels of GAD65, IA-2 and insulin autoantibodies, we nexttested if they were related to Ljungan virus antibody levels. In thediabetic Group B bank voles, antibody levels against Ljungan virusantigens correlated with levels of GAD65 (P<0.0001), IA-2 (P<0.0001) andinsulin (P<0.03) autoantibodies. These associations suggest that Ljunganvirus infection may induce an immune response that will also includebeta cell autoantigens. Without being bound to any one theory, thereseems to be two possibilities. The first possibility is that beta celldestruction is leading to autoantigen presentation in draining lymphnodes; the second possibility is that autoantibodies are formed due tomolecular mimicry between virus and the autoantigen. The latterhypothesis was tested by comparing the predicted amino acid sequences ofthe Ljungan virus CDNA with those of GAD65, IA-2 and insulin.

[0094] Ljungan Virus Molecular Mimicry to Islet Autoantigens

[0095] The comparison between the Ljungan virus amino acid sequencepredicted from the cDNA and the GAD65, IA-2 and insulin sequencesrevealed several potential regions of sequence similarities (FIG. 5a).We searched 1514 picornavirus proteins in Genbank's viral taxonomy atNCBI using stand-alone BLAST and found that these homologies wereexclusively found in parechoviruses (echovirus 22/23) isolates (data notshown). The regions indicated for GAD65, 237-241 and 449-452 have beenimplicated in the middle and C-terminal GAD65 autoantibody binding sites(Schwartz et al., J. Mol. Biol., 287, 983-999,1999). While the 561-569is outside, the 964-976 sequence is within reported autoantibody bindingsites for type 1 diabetes associated IA-2 autoantibodies (Leslie et al.,(supra), 1999). The most interesting significant relationship wasbetween the Ljungan virus antigen and the 45-54 insulin since the 45-54homology maps to the insulin active site (Steiner et al., Diab. Care,13, 600-609,1990). Since antibody levels against Ljungan virus in vitrotranslated antigens correlated to levels of both GAD65 and IA-2autoantibodies in the diabetic Group B bank voles and because of thesignificant sequence similarities (FIG. 5a), we next tested whetherLjungan virus antisera would immunoprecipitate any of the isletautoantigens. While labeled mouse GAD65 was not recognized, the mouseLjungan virus polyclonal antiserum 87-012 (Niklasson et al., (supra),1999) was capable of immunoprecipitating human GAD65 (FIG. 5b),indicating significant epitope specificity (Hampe et al., (supra),2000). The Ljungan virus in vitro translated antigens immunoprecipitatedby the 87-012 Ljungan virus antiserum was reciprocally displaced by bothcold Ljungan virus in vitro translated antigens and human GAD65 but notby human proinsulin (FIG. 5c). The human type 1 diabetes GAD65antibody-positive serum #591 showed concentration dependentimmunoprecipitation of Ljungan virus in vitro translated antigens andboth human and mouse GAD65 (FIG. 5d). Cold Ljungan virus in vitrotranslated antigens but not GAD65 nor proinsulin, displaced theimmunoprecipitation of Ljungan virus in vitro translated antigens by thehuman serum (FIG. 5e). These observations support the possibility ofantibody cross-reactivity due to molecular mimicry between Ljungan virusantigen and GAD65 in mice inoculated by Ljungan virus.

Example 2

[0096] Individuals Infected with Ljungan Virus are Susceptible toDeveloping Diabetes

[0097] We tested to see if new onset type 1 diabetes children hadLjungan virus antibodies by both standard immunofluorescence andradioligand binding assay with Ljungan virus cDNA in vitro translatedantigens.

[0098] Children with Type 1 Diabetes have Ljungan Virus Antibodies

[0099] The commonly used indirect immunofluorescence virus antibody testwas first compared to the radioligand binding assay for Ljungan virusantigen antibodies (FIG. 6). There was a significant correlation(Spearman Rank Sum correlation) between the two assays (P<0.001).Compared to the 17 healthy control children, the children with new onsetType 1 diabetes had increased levels of Ljungan virus antibodies scoredin the immunofluorescence assay (P<0.00l) (FIG. 6). These data indicatethat children with new onset diabetes may have been exposed to Ljunganvirus.

Discussion

[0100] The examples provide evidence that wild caught bank voles maydevelop type 1 diabetes associated with specific beta-cell destruction,insulitis and autoantibodies to GAD65 and IA-2. Our observation that itwas possible to detect Ljungan virus antigen in affected pancreaticislets showing gradual destruction and end-stage fatty degeneration alsoindicates that this virus causes or at least contributes to the loss ofbeta cells. In addition, the diabetic bank voles had increased levels ofantibodies to Ljungan virus cDNA in vitro translated virus antigens. Thelevels of these antibodies were also found to correlate to the levels ofautoantibodies to both GAD65 and IA-2. These data indicate that diabetesobserved in both captured bank voles and in bank voles born to captiveanimals represents type 1 diabetes. Although the histology of the isletsin diabetic voles may be consistent with an acute lytic effect andpresence of viral antigen, it cannot be excluded that beta cells mayalso have been lost by an ensuing T cell or antibody-mediated cellulartoxicity. Also consistent with type 1 diabetes were our observationsthat islet beta cells were specifically lost. The prominentvacuolization or fatty infiltration seems unique to the bank volesdiabetes and differ from other virus causing diabetes in rodents whereinsulitis is seen more often. In particular, the rare occurrence ofislets infiltrated with mononuclear cells suggest that the bank voleislet lesion is less associated with insulitis compared to otherdiabetogenic virus (Jun et al., (supra), 2001; Rayfield et al.,Diabetes, 27, 1126-1140, 1978; Vaaralae et al., (supra) , 1999).

[0101] Diabetes in bank voles was first described during a study ofstereotypic behavior in bank voles (Schoenecker et al., Appl. Anim.Behav. Sci., 68, 349-357, 2000) When captured in the wild, brought tothe laboratory to be kept in standard laboratory mouse cages, and fedlaboratory chow, bank voles developed polydipsia and glucosuria.Diabetes was detected in 4/101 Group A animals that were euthanized inthe forest at the site of the trap which was different from the 22/67bank voles kept in the laboratory. Our data suggest that the diabetessymptoms in these animals fulfill current classification criteria forautoimmune type 1 diabetes in humans (Mellitus et al., Diabetes Care,20, 1183-1197, 1997). The diabetic bank voles sustain hyperglycemia,ketonemia, ketonuria, hyperlipidemia and weight loss, all criteria thatare consistent with type 1 diabetes classification. In addition, thediabetic bank voles had increased levels of both GAD65, IA-2 and insulinautoantibodies. These autoantibodies predict type 1 diabetes in humans(Verge et al., Diabetes, 45 , 926-933, 1996); Bingley et al., DiabetesCare, 22, 1796-801, 1999) and confirm the type 1 diabetesclassification.

[0102] The short period of time it took for bank voles to developdiabetes after capture, and the complete islet beta cell destruction indiabetic voles associated with positive immunostaining for Ljungan virusantigen suggest that acceleration of an existing, low-level viralinfection may induce disease. While the exact mechanism of such aprocess remains unclear, our initial studies of diabetic bank volesindicate that stress is involved in diabetes development. Earlyexperimental stress may lead to increases in adult adrenocortical stressresponses (King et al., Horm. Behav., 36, 79-85, 1999) and such stressresponses may act as a stimulus of virus replication in beta cells. Thisspeculation is supported by the observation that stress induced byswimming increased the frequency of diabetes in our wild caught bankvoles (data not shown). Stress has also been implicated in human type 1diabetes since negative life events increased the risk for childhoodtype 1 diabetes (Thernlund et al., Psychological Stress and the Onset ofIDDM in children, 18, 1995; Hagglof et al., Diabetologia, 34, 579-83,1991; Dahlquist et al., Diabetologia, 34, 757-762, 1991). Similarrelationships could be relevant to our bank voles and therefore aid inunderstanding the etiology of diabetes.

[0103] It is now possible to identify other mechanisms that induceLjungan virus-associated diabetes in bank voles and to conduct studieson intervention and protection, for example by anti-viral agents orreducing responses to stress.

[0104] It has also been found that very high Ljungan virus antigenantibody levels are observed in some of the non-diabetic Group A bankvoles. This may reflect a neutralizing and protective Ljungan virusimmune response with implications for future vaccination approaches.

[0105] Taken together we have demonstrated, first, that bank volesdevelop diabetes that fulfills the criteria for type 1 diabetes:diabetic animals showed persistent hyperglycemia associated with weightloss, ketosis and hyperlipidemia (data not shown) as well as insulindependence associated with specific beta-cell destruction and insulitis.Second, diabetic voles had increased levels of autoantibodies to GAD65and IA-2, and that these autoantibodies correlated to Ljungan virusantigen antibodies. Third, the association between Ljungan virus andbank vole diabetes was supported by the presence of Ljungan virusantigen detected by irnmunocytochemistry in affected diabetic bank voleislets. Fourth, there was significant molecular mimicry between theLjungan virus polyprotein and GAD65, IA-2, and insulin isletautoantigens, illustrated by GAD65 cross reactivity of high titer mouseand guinea-pig Ljungan virus antisera. Finally, a relationship betweenLjungan virus infection and human type 1 diabetes was indicated byincreased levels of Ljungan virus antibodies in children with newlydiagnosed type 1 diabetes.

Example 3

[0106] Data on Diabetes Mellitus in other Mammals than Bank Voles

[0107] 1. Field Voles (Microtus agrestis)

[0108] The field vole develops clinical diabetes with symptoms ofpolydipsia and polyuria identical to bank voles. Ljungan virus has beenisolated from field voles with type 1 diabetes trapped in Sweden. It islikely that field voles are just as good an animal model for type 1diabetes as the bank vole.

[0109] 2. Cats

[0110] Between 1 in 50-500 will develop the disease in a lifetime. Theclinical disease mimics in some animals type 1 diabetes and in someanimals type 2 diabetes. Amyloid deposits localized to the islets ofLangerhans are typical of type 2 diabetes mellitus. However, diabeticcats most commonly have pancreatic islet destruction associated withpancreatic amyloidosis and are insulin deficient like type 1 diabetes.The disease occurs in all ages of the cats but the majority of diabetesaffect older cats (Westermark et al., PNAS USA, 84, 3881-5, 1987;Johnson et al., Veterinary Pathology, 22(5):463-8, 1985; and Yano etal., Veterinary Pathology, 18(3) : 310-5, 1981). We have investigatedthe pancreas tissue of diabetic type 1 and type 2 as well as normal catsusing immunohistochemistry (IHC). As in the bank voles Ljungan viruscould be detected in the destroyed islets of the pancreas in thediabetic type 1 and 2 cats but not in the normal cats (Bo Niklassonunpublished observations). This is very strong evidence suggesting thatLjungan virus cause diabetes in cats.

[0111] 3. Dogs

[0112] Between 1 in 50-500 will develop the disease in a lifetime. Areview of the literature has shown that over half of the documenteddiabetic dogs are type I diabetes. However, type 2 is also common indogs with obesity (Stogdale L. Cornell Veterinarian. 76(2):156-74,1986). We have investigated the pancreas tissue of diabetic type 1 andtype 2 as well as normal cats using immunohistochemisty (IHC). As in thebank voles Ljungan virus could be detected in the destroyed islets ofthe pancreas in the diabetic type 1 and 2 dogs but not in the normaldogs (Bo Niklasson unpublished observation). This is very strongevidence suggesting that Ljungan virus caused the disease.

[0113] 4. Guinea Pigs

[0114] Spontaneous diabetes mellitus in guinea pigs, parallels in manyways the syndrome known as juvenile diabetes mellitus in man: elevatedblood glucose levels; reproductive dysfunction in the female;degranulation and severe cytoplasmic vacuolation of beta cells, severefatty degeneration of acinar cells, and hyperplasia of the islets of thepancreas; and a high frequency of abnormal pancreatic secretions (Langet al., Diabetes, 25(5):434-43, 1976). The severity of pathologicchanges in the pancreatic islets parallel, in general, the severity ofthe clinical symptoms. The other clinical parameters of note areelevated serum triglycerides, normal serum but elevated aorticcholesterol, and absence of ketonemia or ketonuria. Microangiopathy,another characteristic of juvenile diabetes mellitus in man wasdemonstrated as a significant increase in the thickness of the basalmembranes in peripheral capillaries. A glomerular lesion encountered insome of the diabetic guinea pigs was shown to be similar to theglomerular sclerosis seen in human diabetics. Although a definitiveetiologic agent was not identified, the disease was clearly contagiousin origin.

[0115] 5. Rabbits

[0116] Spontaneous diabetes mellitus has been observed in a female NewZealand white rabbit. Three groups of animals could be identified. Someanimals had overt diabetes characterized by fasting hyperglycemia anddepressed intravenous glucose stimulated serum insulin levels (Conawayet al., Clinical & Experimental, 30(1):50-6, 1981). This abnormality isseen between 1 and 3 years of life. Another group of animals developedabnormal glucose disposal with normal or slight elevations in fastingserum glucose levels. Glucose stimulated insulin levels are alsosignificantly lower in the rabbits with abnormal glucose disposal. Theremaining animals exhibit no apparent abnormalities of glucosemetabolism. Despite marked increases in serum and urinary glucose, onlymild ketonemia was observed. The relatively late onset of diabeticsymptoms, lack of obesity, severe hyperglycemia, and depressed insulinsecretion without ketoacidosis make this a model with many of thecharacteristics of insulin responsive diabetes as seen in non-obesehuman adults.

[0117] 6. Hamster

[0118] Chinese hamsters spontaneously develop diabetes mellitus andcardiomyopathy. The diabetic hamsters, shows body weight loss,hyperglycemia (mean fasting plasma glucose 402 mg/dl), hypoinsulinemia,hyperlipidemia and ketonemia. The diabetic hamsters showed reducedactivities of cytoplasmic glycolytic key enzymes: hexokinase, pyruvatekinase and phosphofructokinase; increases in cardiac glycogen andglucose-6-phosphate contents; and a 40% decrease in cardiac ATP content,indicating decreased energy production. An accumulation of myocardialtriglyceride and cholesterol was found in the diabetic hamsters (Eto etal., Diabetes Research & Clinical Practice, 3(6) :297-305, 1987).

[0119] Syrian hamsters infected with rubella virus passaged inbeta-cells also develops diabetes that closely parallels the diabetesobserved with congenital rubella (Rayfield et al., Diabetes, 35(11):1278-81, 1986). The hamsters develop hyperglycemia and hypoinsulinemia,which are sustained throughout the 15 week study period. A mononuclearinfiltration of the islets, isolation of rubella virus from wholepancreas, the presence of viral antigen in beta-cells byimmunofluorescence localization, and cytoplasmic islet cell antibodies(40%) are demonstrated. These data suggest that an autoimmune processand diabetes develop after rubella virus infection in neonatal hamsters.This model may uncover the precise mechanism by which rubella virusinduces similar disease in humans.

[0120] All documents referred to in the above description areincorporated herein by reference.

1 12 1 7604 DNA Ljungan virus 87-012 1 tttgaaaggg gtctcctggt ggggtgggtacacttctcgc tcgatgagtg ggggtgtggc 60 tcattgccca cacctggttg gttcccaggttcatacaata accatcaata aacttttaac 120 atctaagata gtattatccc atactagactggacgaagcc gcttggaata agtctagtct 180 tatcttgtat gtgtcctgca ctgaacttgtttctgtctct ggagtgctct acacttcagt 240 aggggctgta cccgggcggt cccactcttcacaggaatct gcacaggtgg ctttcacctc 300 tggacagtgc attccacacc cgctccacggtagaagatga tgtgtgtctt tgcttgtgaa 360 aagcttgtga aaatcgtgtg taggcgtagcggctacttga gtgccagcgg attaccccta 420 gtggtaacac tagcctctgg gcccaaaaggcatgtcattt gaccactcag gtacacaacc 480 ccagtgatgc acacgcttag taatggcttagtaacaaaca ttgattgatc atttgaaagc 540 tgttaggagg tttaggtatg acgggctgaaggatgccctg aaggtaccca taggtaacct 600 taagcgacta tggatctgat caggggcccaccatgtaaca catgggtaga agtcttcgga 660 ccttgggtta aaaaacgtct aggccgccccccacagggat gtggggtttc ccttataacc 720 ccaatattgt ataatggctg catccaaaatgaatcccgtt ggcaacctgc tctccacagt 780 ctcctcaacc gttggatctc ttctacaaaacccctctgtt gaagaaaagg aaatggattc 840 tgaccgtgtt gctgcctcta ctacaaccaatgctggtaat ttggtacaag cttctgtggc 900 cccaactatg cctgtcaagc cagattttaaaaacacagat gatttcttgt ccatgagcta 960 ccgttcaaca acagccccaa ccaaccccacaaaaatggtt cacttggcac atggcacttg 1020 gacaactaat cagcacagac aggcattggtcgcatcaatt actctaccgc aggcattttg 1080 gcccaatcaa gattttccgc atgggggcagtctcgttatt ttgcagcagt gcgctgtggc 1140 tttcatattc aagtacagtt gaatgttaacatcggttctg ctggttgctt gattgccgcg 1200 tacatgccaa agacggccca tgatcatatgaacacctata cttttggttc ttacaccaac 1260 ctgccacatg ttttgatgaa tgcggcaacgacatcccagg ctgatctcta tataccctat 1320 gtttttaatc ataactatgc aagaactgattcagatgatt taggaggcat ttacatttgg 1380 gtttggtcag ctctcacagt tccatcaggttcacctacaa cagtggatgt gaccattttt 1440 ggttcattac tcgacttgga ttttcagtgtcctcgtcctc ctggtgcaga cacagtaatt 1500 tacacacaag ggaaaagaac tgttcgaaagaccaagacat caaagttcaa atgggtcagg 1560 aataaaattg acatagctga aggtccaggagcaatgaaca tcgctaatgt tctctccaca 1620 actggtggtc aaactattgc cttggttggtgaaagagcat tctatgaccc aagaacagct 1680 ggtgctgcag tgaggtgcaa ggacctcatggagatcgcca gaatgccaag tgtgttctta 1740 ggagagagta ctgaaccaga tggtcgaaggggctatttca cctggtcaca tacaatctca 1800 cctgttaatt gggtttttga tgaccacatttatttggaaa acatgcccaa tctgagatta 1860 ttctcctcct gttacaacta ttggaggggctcctttgtta ttaaactgac agtttatgcg 1920 tcaactttca acaaaggacg cttgaggatggcatttttcc caaacagaga aggcgcctac 1980 acacaggatg aagctcagaa cgcaatctttgttgtgtgtg atataggctt gaataatacc 2040 tttgagatga ccatccccta cacttggggcaactggatga gaccaacaag aggaaactcc 2100 ttgggacatc tgaggattga tgtgttgaaccgtcttacat acaacagttc ttccccaaat 2160 gcagtcaact gcattcttca gattaagatgggggatgatg caatgttcat ggtgcctacc 2220 acatctaatc tagtttggca aggtctgcactcatggggtt cagaaatgga cttggtggac 2280 tctctcgaca atccagacga gatacaagacaatgaggaaa tacaaaccca aaatgtggag 2340 gctgcacaag gggaagaagc tgcgactgaagttggtctta gggcaacaga aaatgatggc 2400 agtctttcag aacaattgaa tatgagtcaacccatgttcc tgaattttaa gaagcataaa 2460 gtcaacatct atgcagcatc ccataccaaagttgatcata tttttggaag agcttgggca 2520 gtgggggttt ttaacacaga aacagctgccatacaaaaat ttgatttgca ctttccaact 2580 tctactcatg gtgcattgtc tagatttttctgcttctgga ctggagagtt aaatattcac 2640 attttgaatg tgtcaaccac aaatgcctttttgaaagttg ctcacacatg gtttggcact 2700 gattctggaa ttgcccggac agctactttggaatcaaatg gaacaatgat tataccacca 2760 aacgagcaaa tgacactttg tgtaccctattattctgagg ttccattaag atgtgttaaa 2820 ggttcagaca ggaattcagc cggacttggttctctcttca cacaggctgt gggcagaaca 2880 atctctaatc gggtacaaat ctttgtgagcttccgctgtc ctaatttttt cttcccacta 2940 cctgcgccca gggaagccac gtctcgaagcatattggaac gagtggatga agcaaatgcg 3000 gaagaacttg aagctgtctt ggaagctagaacaccagatg caccgctccg cctcaagttt 3060 aatccagaag atcctttgaa acaattgcgggaggcggcca aggcttactt taatataatg 3120 cacagtgatg aaatggattt tgccggggggaaatttttga accaatgtgg tgacgtggaa 3180 accaacccag gccctgacat tgagttggtctataaaaaca gaggcttcta taagcattat 3240 ggagttagat ttggtggtca tatctaccacttgaattcac aagacattct gtcaaccgca 3300 attacaggca agtctgactt cattaaggaagaagatgatg gcaaatgggt tcatgctatg 3360 acagcaccat tggactactt tactgaaaagtacatcaatt caatggttgg ctccaaacac 3420 atcttttccg ccacctccaa ttgtgagaccattgccagag atcttttccc agggagaaag 3480 gaaataactc agtccaaagc cttggggattattggggtca tcttgttgtc agcctctctt 3540 ctttcattgc ttgctgtacc ctgggattattcctcacttc aaactgttta taaccaatcc 3600 attgaaggtg atgcttctgg cctcacactcctaagtcaaa gatgcatgac ttttttttcc 3660 aatacaatgt gtgaaacttt taataatgatcttgttaagt ttattattaa gattttggtg 3720 cggcttttgt gctacatcgt tctttattgccatgcaccaa atatgctgac aaccatgtgt 3780 ctgggaactc ttcttgtttt ggacattacaacttgtgaaa tcttgtctgc taacaccaaa 3840 gcactctttc aggcattggt tgatggtgatgtgaagagtc ttgtctggaa aattgctgag 3900 aatatgcaat ttgcccaatc caaggatgaacaagctgagg acatggcagc aaccttcaac 3960 tttgcctctg acatggttaa ttttgtgccaatggaacaga tgagacaaga aggctggaga 4020 gaatttaatg atgtttctat gtcctttcggcatgttgaat ggtggctgac tatgttcaaa 4080 aaggtgtaca atgttctgaa aagtatttttgctcctagta ttgaacagaa ggctgttgat 4140 tggatagatc gcaatcaaga gtacattgccgatgttttgg accatgcttc caacatcatc 4200 ataaaaatga aggatccaaa agaacagggagagcatcaac cattagtgaa tactttgagg 4260 ttttgaaaca actaaagcca attgtgtccctttgcatgaa ggttgccccc tccactaagt 4320 tttcctctca agtgtttaga atctattctgaaatgatgag ggtcaatgtt agagtgcctg 4380 cgaatactga cttgactaga ctggaacccattggcatttg ggtttctagt gagccaggac 4440 agggtaaatc attctttaca catatgttgagtacctgtct tctgaagtcc tgcaatttag 4500 aaggaattta taccaacccc acaggttcagaatttatgga tggctatatt gggcaggaca 4560 ttcatatcat agatgatgca gggcaaaatagagaggaaaa agatttggcc ttgttgtgtc 4620 agtgtatttc ttctgtgcct tttactgttccaatggcaga tttgacagag aagggcactt 4680 tttatacaag caaaattgtg attgccaccaccaataaatt tgattttacc tcaatggttt 4740 tgacagatcc agcagccctt gaaaggagatttccgttcca cttgcgcatt agagctgtag 4800 ccagctattc gcgcaacaac aaactagatgtggcccgttc aatggcggcc atggcagatg 4860 gttcttgctg ggaatattcc acagatggtggtagggcttg gaagaccctc tccatggatg 4920 aacttgtgaa acaaatcacg gcagtttacacacagaggtc agatgccctt atggtttgga 4980 aaaggaagtt gaataccatc aggaacgaaatgagccctgg atcatccacc ggcaggattt 5040 ttgaaccctt agaggaaaca ctctgtgctttggaacgtcg ctttggtcaa cttgctgata 5100 gtcttaaaga caactatcat aaaacagctgatgagttgat tgaagctata gaagatatga 5160 tggcaccgtc acagagccct tttgcatgctttgctgaatc ctatcgaccc accattaaat 5220 atactgccag tgataaagtt aaatcatgggttaaaaatca tatgaataga tggaaagaat 5280 ttgtaatgag aaataaaggc tggtttacacttttttctgt gctctcatca tttctctcga 5340 ttcttactct tgtctattta cactataaaaaggagaagaa agaggaagag agacaggagc 5400 gggcttacaa ccctcaaact gcaatttctaagaagggggg taagcctaag ctctcattag 5460 tgaaaaccac aaactttgtt aatgaagcaccctatatgca agatcttgaa cattgctttg 5520 cacaaacggc ctacatttct tctccagaaacccaagatat aatacattgt gctgccttga 5580 gtgaagacac cattttggtt tatggacattctcagtttta ttttaaccgc tatgaggacc 5640 tgcggttaca ttttaaaggg gccatttttcccatagaagg gggaaaaatt tctcaagtta 5700 ccgtgaatgg acagcctatg gatttgatacttgtgaagat agataaactt ccaataacat 5760 ttaaaaatta tacaaaatat tatacaactgaggttggcaa ggaaactctt ttaatttgga 5820 attcagaaaa gggcaggttg gccatgcctgttcaatgtgt ggctccggct ggtccggtgg 5880 agacaatgga aggaacaatt actcataagacctattcata taaagtggca tcaaaaaaag 5940 gaatgtgtgg gggccttttg gtcactagagtgcatggcac attcaaggtt ctgggaatgc 6000 acattgcagg caatgggcaa gttgcacgagctgcagcagt tcactttata tccaatggtg 6060 cagctggctt tatggatcaa ggtgttgttgtggccaaaga aaagttacaa aagcccattt 6120 atttgccatc taagacagct ttgaatcccagtcccttgaa tggtgtagtc cccgtgaaaa 6180 tggaaccagc tgtgcttagt cctcatgacaccaggcttga agtcatcatg cccagcgttg 6240 tgaaaacagc ggcagctaag tatagagttaatatttttaa tcctgatttt gagatttggg 6300 agagagtggt ggatgagtta aaatcaaagtttagaaccaa acttggaatt cataaacatg 6360 tctcttttca gaaagcagtt cagggtttctcctccctttc atctcttgat ctttccacat 6420 ccccaggaca aaagtatgtt gaaaagggtatgaagaagag agatcttttg tccactgagc 6480 cattttggat gcatcctcaa ttggaaggtgatgttaaaga tatacttggg gccgtttact 6540 ctggtaaaaa gccccataca ttttttgctgcacatttgaa agatgagttg cgcaaaaaag 6600 aaaagattgc gcaaggaaag acccgctgcattgaagcctg ttcaattgac tatgtgattg 6660 cctacagagt tgtaatgtcc tcactctatgaggcaatcta tcaaactccg gctcaggagt 6720 tgggcctggc agtgggaatg aatccctggacagattggga tccaatgatc aatgttttgc 6780 agccatataa ctatggcctg gattactcatcttatgatgg cagcctttct gaacaactga 6840 tgagatatgg agtggaaata cttgcctactgtcatgaaca accagaggct gtaatgattc 6900 ttcatgaacc tgttataaac tctcaacaccttgtgatgga tgaaatctgg catgtgaatg 6960 gtggaatgcc ctcaggggcc ccatgtacaactgtgctaaa ttccatatgc aatctgctag 7020 tttgtacata tttggcctat gagcagagcttggatattga ggtgttaccc attgtttatg 7080 gagatgatgt aattttttct gtttcatcccctttggatgc tgaatacttg gttcagagtg 7140 cagcccaaaa ttttggaatg gaagtgacctcatcagataa atctggtccc cctaaacttt 7200 tgaaaatgga tgagattgaa tttttaaagaggacaacaaa attttttcct ggctccacct 7260 acaaggtggg ggccttgagc ctggataccatggaacaaca tattatgtgg atgaagaatt 7320 tggaaacctt tccagaacaa cttgttagttttgagaatga attggtgttg catgggaaag 7380 aaatttatga tgattataaa aataggtttaatcctatttt gaatcaatgg cgagtgtgca 7440 tgcaggacta tgaagtggcc ctgcatcgcatgctacgcta tgtttttgat tgaattgatt 7500 tagtttgatt ttgattttat tagctttagtttatgtaagt tagaattaga ttattttagt 7560 ttagttttaa agattttgat ttgattgaatttggcccacc aatc 7604 2 7609 DNA Ljungan virus 145SL 2 tttgaaaggggtctcctggt ggggtgggta cgtctctcgc tcattgagtg ggggcgtggc 60 tcaccaaccacacctggttg gtccccaggt tcatgcaata accacttttt gtaatcttta 120 catctaagcttaattcaccc actagaactg gacgaaaccg cttggaataa gtttggttct 180 ctcttgagtgtgttttgtgt tagcataatt tctgtctcta gagtgcttta cactctagta 240 ggggctgtacccgggcggtc ccactcttca caggaatctg cacaggtggc tttcacctct 300 ggacagtgcattccataccc gctccacaat agaagatgat gtatatcttt gtttgtgaaa 360 tgctcatgaaacgtgtgtgt aggcgtagcg gctacttgaa tgccagcgga acccccctag 420 tggtaacactagcctctggg cccaaaaggc atgtctctga ccattcaagt acacaacccc 480 agtgatacacacatttagta atggctcagt aatggacatt gattgatcat cagacaattg 540 ttaggaggcctaggtatgac gggctgaagg atgccctgga ggtacccgca ggtaacctta 600 agagactgtggatctgacca ggggcccacc atggaaacat gggtagaagt cttcggacct 660 tgggttaaaaaacgtctagg cccgcccccc acagggatgt ggggtttccc ttataacccc 720 aatatcacattatggctgca accaagatga atcccgttga gaatcttctt tctactgtct 780 cctccaccgttggctcactg ctacaaaatc ccaccatgga agaaaaggaa atggactcag 840 atcgtgttgcggcatccacc actactaacg ctggaaatgt agttcaggct tcagttgccc 900 ccaccatgccaattaaacca gatttcaaga acacggataa ctttttgtca atgagttata 960 gcccaaatactgcacctaca aatccaacaa aaatggtaca tttggctaat ggaacatgga 1020 ctacatcgcagcatcgacag tctttggttg catcgattca gctaccacag gcattttggc 1080 ccaatgaacgctatccggct tggggtcaat cacgctattt tgctgcagtc cgatgtggct 1140 ttcatattcaggttcaattg aatgttaaca ttggctcagc aggttgtttg atagctgcct 1200 atatgcccaaaagtgcacat gatcatatgg atacatatac atttagttcc tacaccaatt 1260 tgcctcatgttctgatgaat gctgccacca cgtctcaggc tgatttgtat ataccctatg 1320 tgcataatcataattatgca aagacagatt cagatgactt gggtggtata tacatttggt 1380 gttggtctgccctcacagtt ccatcaggtt ctccgacaac tgttgatgtc acaatttttg 1440 gctccttgcttgacttggac ttccagtgcc ctagaccacc aggtgctaat actgtcatat 1500 ttacacaaggcaaaagaact gccaggaaaa ccaaagcaac aaaatttaaa tggacaagga 1560 ataaaatagacattgctgaa ggtcctggcg ctcttaatat tgccaatgtc ttgtctacta 1620 cagggggccaaactgttgcc ctcgttgggg aaagagcttt ctacgatccc agaactgcag 1680 gagccgctgtgcggtgtaag gatttgatgg aaattgccag aatgccatca gtctataagg 1740 gggagagaactgaacctgga ggaactaatg gctattttca atggtctcat acgcactccc 1800 ctataaattgggtttttgac gggggaattc atttggaaga catgcccaat ctaaatttgt 1860 tttcctcatgctataactat tggagaggct caattgtttt gaaactcact gtgtatgcat 1920 caacctttaacaagggtaga ttgagaatgg ccttcttccc aaatcatgat gcaaggtaca 1980 cagaggaagaagcacaaaat gccatcttca tggtgtgtga tattgggctc aacaacactt 2040 ttgaaatgaccatcccatac acctggggaa actggatgag accaactagg ggatctgtca 2100 ttggatggcttaggattgat gttttgaatc gcctcactta taacagttcc tcacccaatg 2160 ctgttaattgcattcttcag gttaaaatgg ggaatgatgc caaatttatg gtacccacca 2220 catctaacattgtgtgggaa ggtctccact catgggggtc tgagatggac ttactggaca 2280 gtttggataatccagaagag attcaagata tggaggaacc agaatctgaa aatgtggagg 2340 ccgcacaaggagaggaagcc gccactgccg ttggccttcg agccaccgaa aatgatggat 2400 ccctatctgaacaacaaaac atggcacaac caatgttttt gaattttaag caacatagag 2460 tggacatttactctgcttcc cacaccaaag ttgaccatat ttttggtagg gcgtgggcag 2520 tgggaatttttaatgtgact aatgctaata tatccaaatt tgaccttaac tttcccacaa 2580 ccacacatggtgcattgtgt cgcttcttct gtttctggac gggagagctt aacttgcata 2640 ttttgaacatttcttcttcc aatgctccag tcaaagttgc tcacacatgg tttggcacag 2700 attcaggcattgccaggact gcaactttgg aatcaaacgg ggttatcatc ataccaccaa 2760 atgagcaaatgacactctgc ataccctatt attctgaggc accattgcgc tgtgttaagg 2820 ggccacattcagctggtgct ggattgggct caattttcac acagtgtatt ggcaacagcg 2880 ttaataacaggattcaaatt tttgttagtt ttcgctgccc aaacttcttt tttccccttc 2940 ctgcaccccatgaggcttct tcaaggtcaa ttttgcagag aatttccact gctagcgcag 3000 atgagttagaagctgtcttg gacgcaaaaa cacctgatgc tcctgtgcgc ttgtgctacc 3060 aaccagaggatcctttgaga caacttaggg aggcagctaa ggcatatttc aatattatgc 3120 acaatgatgagatggactat tctggaggta aattcttgaa tcagtgtggt gatgtggagt 3180 ccaatccaggtcctgatatt gaattagtct ataagaacag aggcttttat aaacattatg 3240 gggttaggtttggtggcttt atttaccatc ttaattcaca agacattttg tcgacagcca 3300 tcactggaaaatcagacttc ataaaagagg aagatgatgg taaatggaca catgctatga 3360 ctgcacccctggattatttt actgagaagt atgtgaaatc aatggttggt tcaaaacaca 3420 ttttttccgccacatcaaat tgtgaaacca ttgccaggga tttgtttcca ggaaagaagg 3480 agattagtcaatctaaagct ttgggtatta ttggtgtgat ccttctttct gcatctcttt 3540 tatccctacttgccgttcca tgggattatt cctcacttca gacagtttat aatcaatcaa 3600 ttgaaggggatgcttcaggc ttaacacttt tgagccagag atgcatgact tttttttcca 3660 ataccatgtgtgaaactttt aataatgatc ttgtgaagtt tataattaag attttagtta 3720 ggcttctttgctatattgtt ctttattgtc atgcccctaa tatgcttaca acaatgtgtt 3780 taggcacccttttggttttg gatattacca catgtgagat tttatcagcc aatacaaagg 3840 ccctgtttcaagctcttctt gatggagacg tcaagaattt ggtttggaag attgcagaga 3900 acatgcagtttgcccagtct acagatgagc aggcagagga aatggctgcc accttttcat 3960 ttgccaaagacatggttgac attcatccaa ttggggctga gccatttcaa aaccaaggct 4020 ttagggagtttaatgatgtg tcaatgtcct ttcgccacat tgaatggtgg cttacaatgt 4080 ttaagaaagtttacaatgtt cttaagggca ttttctctcc atccattgag cagaaagcgg 4140 tggcgtggttggatcgcaac caagaatatg ttgcatcaat cttagatcat tgctctgaca 4200 tgattatccgcatgaaagac ccaaaacaac agcggaaccc caagaccatt gaagaatatt 4260 ttgatgtgttaaagaaaatg aagcccttgg tgtcactctg cattaaagtt gccccgtcaa 4320 caaagttttcatcccaagtg tttaggttgt attcagagct aatgaaggtt aatgttagag 4380 tgccggttaacacagatctc acacgcattg agccaattgg tgtgtggatc tccagtgagc 4440 caggtcagggaaaatctttc tttactcaca tgcttagcac ttcacttttg aaaagttgta 4500 atttggatggggtgtatacc aatgccacag gctctgagtt tatggatgga tatgttggtc 4560 aagatatacacattattgat gatgcaggac aaaatcggga agagaaggat ttggctctgc 4620 tgtgccagtgcatctcatct gtgccattta ctgtacctat ggctgatcta acagagaaag 4680 ggacattttataccagcaag attgttattg ccacaaccaa caagagtgat ttcaattgca 4740 tggttttgacagatccagct gctctagaga ggcgtttccc atttaatttg agaattaggg 4800 cagttaaaagttttatgaat aaggacagaa agttggatgt gccaagatca atgggagcca 4860 tggcagatggatcctgctgg gagtgctcta tggactatgg cagaacctgg aacaccgtgg 4920 tgatgagagatcttgtgaaa caaataacag aaatgtataa acaaagagat gatgccctga 4980 ctgtttggaagtataagtta aatcagatta ggaatgagat gtcccctggt gactcaattg 5040 gccgcattctcgatccaatg gaggagacac tctgttcatt ggagcgcagg tttggccagt 5100 tggcagatagtcttagagaa aattaccata ggacagctga tgaactaatt gaagttatag 5160 aagacatgatggcaccaggg aatagtccct ttgcatgctt cgaaagtgta gcaccatcac 5220 ttaaaccaagaacagcttgt caaaaagtta aagattgggt aaaacaacac atgattagat 5280 ggggcaactttgtgatgagg aataaaggct ggtttacact tttttctgta ctttcatctt 5340 ttctttcaattcttactctt gtttatttac attataaaaa agagaaaaaa gaggaagaaa 5400 gacaagagcgggcttacaac cctcaaactg caactcccaa gaaggggggt aagccaaagc 5460 tctctttggtaaaaactaca aattttataa atgaggcacc atatatgcag gatttggaac 5520 actgctttgcccaaacagcc tacatttcat ccccagagac tcaggatata attcattgtg 5580 ctgccttgtgtgaggatacc attttggttt atggacattc acaattttat tttaaccgct 5640 atgaagatttgcggttacat tttaaaggag ccatttttcc tattgagggt ggaaaaattt 5700 cacaagttactgtgaatggg cagccgatgg atttgattct tgttaaaata gacaaacttc 5760 ccataacctttaaaaattat accaaatatt acacaactga aattgggaag gaaactcttt 5820 taatttggaattctgagaaa gggagactgg ctatgccagt ccaatgtgtt gccccggctg 5880 gaccggtggaaacaatggaa ggcaccatca ctcataaaac ctattcctac aaagtggcat 5940 caaagaaaggcatgtgcggt ggactcctag ttactagagt gaatggaaca tttaaggttt 6000 tggggatgcacattgctggg aacggacagg ttgcgcgggc cgcagcagtt cacttcattt 6060 caaatggggctagtggtttt atggatcagg gggttgtggt tgcaaaagag aagatgcaga 6120 aaccaatttatttgccatct aaaacagcac taaatcctag ccctttgaat ggtgttgtgc 6180 ccgtgaagatggagcctgca gttcttagcc ctcatgatgt tagacttgaa gtgattatgc 6240 caagcgtggttaaaaatgca gcagccaagt acagagttaa catcttcaac ccagattttg 6300 aaatctgggagagggtggtt gatgaattga aagcaaggtt tcgatctaag cttggcatac 6360 acaaacatgtttctcttcaa aaggctgtgc aaggtttttc ctccctttcg tctcttgatc 6420 tttctacctctccagggcaa aagtatgttg aaaaaggaat gaagaaaagg gatcttttgt 6480 ccactgaaccattttggatg catcctcaat tggaaagtga tgttaaagat atacttgggg 6540 cagtttattctggtaagaaa ccccacacat tttttgctgc ccacttgaaa gatgagttgc 6600 gcaagaaggaaaagattgcg caaggaaaga cccgctgcat tgaagcatgt tcaattgatt 6660 atgttattgcctatagagtt gtgatgtcct ctctctatga ggcaatttat caaaccccag 6720 ctcaagaattgggcttggca gtgggaatga atccctggac agattgggat ccaatgatta 6780 atgttttgcagccttataat tatggtttag attattcatc ctatgatggc agtctttctg 6840 aacagttaatgagatatggt gttgaaatac ttgcttattg tcatgagcaa ccagaagctg 6900 tgatgattctccatgagcca gttataaatt cgcaacacct tgtgatggat gaaatctggc 6960 atgtaaatggaggaatgccc tcaggagccc catgtacaac tgtgctaaac tctatatgta 7020 atttgctggtttgtacatat ttggcttatg agcagagttt ggacattgag gtgttgccta 7080 ttgtttatggggatgatgtg attttttctg tttcttcacc attggatgct gaatatttgg 7140 ttcagagcgctgcccaaaat tttggaatgg aagtgacatc atcagataaa tctggccccc 7200 caaaacttttgaaaatggat gagattgaat ttttaaagag gacaacaaaa ttttttcccg 7260 gctccacctataaggtgggg gccttgagcc tggataccat ggaacaacac attatgtgga 7320 tgaagaatctggaaaccttt ccagaacaac ttgttagctt tgaaaatgag ttggtgttgc 7380 atgggaaagaaatttatgat gattataaaa ataggtttaa tcctattttg aatcaatggc 7440 gagtgtgcatgcaggactat gaagtggctc tgcatcgcat gctacgctat gtttttgatt 7500 agattgatttagtttgattt tgattttatt agttttattt taggttagaa ttagattatt 7560 ttagtttagttttaaggatt ttgatttgat tgaatttggc ccaccaatc 7609 3 7608 DNA Ljungan virus174FL 3 tttgaaaggg gtctcctggt ggggtgggta cactcctcgc tcaatgagtgggggtgtggc 60 tcattgccca cacctggttg gttcccaggt tcatacaata accatcaataaacttctcaa 120 catctaagct actactatcc cacactaaac tggacgaagc cgcttggaataagtctagtt 180 tcattctgtg tgtgttttgc actgaaatta tttctgtctc tggggtgctttacacttcag 240 taggggctgt acccgggcgg tcccactctt cacaggaatc tgcacaggtggctttcacct 300 ctggacagtg cattccacac ccgctccaca gtagaagatg atgtgtgtctttgcttgtga 360 aaagcttgtg aaaatcgtgt gtaggcgtag cggctacttg agtgccagcggactacccct 420 agtggtaaca ctagcctctg ggcccaaaag gcatgtcaat tgaccactcaggtacacaac 480 cccagtgatg cacacgtcta gtaacggctt agtaacgagc attgattgatcatttgaaaa 540 ctgctaggag gtttaggtat gacgggctga aggatgccct gaaggtacccataggtaacc 600 ttaagcgact atggatctga tcaggggccc accatgtact acatgggtagaagtcttcgg 660 accttgggtt aaaaaacgtc taggcccgcc ccccacaggg atgtggggtttcccttataa 720 ccccaatatt gtataatggc tgcatccaaa atgaatcccg ttggcaacctgctttccaca 780 gtctcctcaa ccgttggatc tcttctacaa aacccctctg ttgaagaaaaggaaatggat 840 tctgaccgtg ttgctgcctc caccacgacc aatgctggta atttggtgcaagcttctgtg 900 gctccaacca tgcctgtaaa accagacttt aagaacacag atgacttcttgtccatgagc 960 taccgctcaa caacggcccc aaccaacccg acaaaaatgg ttcacttagcgcatggaact 1020 tggacaacta atcagcacag acaggcattg gttgcatcaa ttaccctaccacaggcattc 1080 tggcccaatc aagattttcc agcatggggg caatctcgct attttgcagcagtgcgctgt 1140 ggctttcata tacaagtgca gttgaatgtt aacattggtt ctgccggctgcttgattgcc 1200 gcatacatgc caaagacggc ccatgatcat atgggtacct atacttttggctcctacacc 1260 aacctgccac atgttttgat gaatgcagca acgacatctc aggctgatctctatataccc 1320 tatgttttta atcacaatta tgcacgaact gattcagatg acttaggaggtatttacatt 1380 tgggtatggt cagctctcac agttccatca ggttcaccta ctacagtggatgtcaccatt 1440 tttggttcat tactcgactt agattttcaa tgtcctcgtc cccctggagcagccacagta 1500 atctacacac aagggaaaag aactgttcga aagaccaaaa catcaaagtttaaatgggtc 1560 aggaataaaa ttgacatagc tgaaggccca ggagcaatga acattgctaatgttctctcc 1620 acaactggcg gtcaaactat tgccttggtt ggtgaaagag cattctatgacccaagaaca 1680 gctggtgctg cagtaaggtg caaagatctc atggagatcg ccagaatgccgagtgtgttc 1740 ttgggagaga gcactgaacc agatggtcga agaggctatt ttacctggtcacatacaatc 1800 tcacctgtta attgggtctt tgatgatcat atttatttag aaaatatgcccaatttgaga 1860 ttgttttcct cttgttataa ttattggaga gggtcttttg ttattaaattaacagtctat 1920 gcatcaactt tcaacaaagg acgcttgagg atggcattct tcccaaacagagagggcgcc 1980 tacacacagg atgaagccca gaatgcaatc tttgttgtct gtgatataggcctgaataac 2040 acttttgaga tgaccatccc ctacacttgg ggcaattgga tgaggccaacaagagggaat 2100 tccttgggac atttgaggat tgatgtgctg aatcgtctca catacaacagttcctccccg 2160 aatgcagtca actgcattct tcagatcaag atgggagatg atgcaatgtttatggtgccc 2220 accacatcta atctagtttg gcaaggccta cattcctggg gttcagaaatggacctggtg 2280 gactcccttg acaatccaga agagatacag gataatgagg aaatacaaactcagaatgtg 2340 gaggcagcac aaggggaaga agctgcaaca gaagttggac ttagggctacagaaaatgat 2400 ggtagtcttt cagaacaact gaatatgagt caacccatgt tcttgaatttcaagaagcat 2460 aaagttaaca tctatgcagc atctcacact aaagttgatc atatttttggcagagcttgg 2520 gcagtaggag tttttaatac agaaacagct gccatacaaa aatttgatttgcattttcca 2580 acttctaccc atggtgcatt atctagattt ttctgttttt ggactggagaactgaacatt 2640 cacatcttga atgtgtcaac cacaaatgca ttcttgaaag ttgctcacacatggtttggc 2700 actgattctg gaattgctcg gacagccact ttggaatcaa atggaacaatgattatacca 2760 ccaaatgagc aaatgacact ctgtgtgccc tattattctg aggtcccattaagatgtgtt 2820 aagggctcag acaggaattc agccggtctt ggttctcttt tcacacaagctgtaggcaga 2880 acaatttcca atcgggttca aatttttgtg agcttccgct gtcctaattttttcttccca 2940 ctacccgcgc ccagagaagc cacgtcccga agcatattgg aacgagtggatgaagcgaat 3000 gcagaagaac ttgaagctgt tttggaagct agaacaccag atgcgccgctccgcctcaaa 3060 tttaatccag aagacccctt gaaacaattg cgggaagcgg ctaaggcctactttaatata 3120 atgcacagtg atgaaatgga ttttgccggg gggaaatttt tgaatcaatgtggtgatgtg 3180 gaaactaacc caggccctga cattgagttg gtctataaaa acagaggcttttataaacat 3240 tatggggtta gatttggtgg ctatatctac catttgaatt cacaggatattctgtcaact 3300 gcaattacag gcaagtctga tttcattaag gaggaagatg atggcaaatgggttcatgct 3360 atgacggcac cactggatta ttttactgaa aagtacatca attcaatggttggttccaaa 3420 catatttttt ccgccacctc caattgtgag accattgcca gagaccttttcccagggaga 3480 aaggaaataa ctcagtccaa agccttggga attattgggg tcattttgttgtcagcctct 3540 cttctttcct tgcttgctgt accctgggat tattcctcac ttcaaactgtttataaccaa 3600 tccattgaag gtgacgcttc tggcctcaca cttttaagtc aaagatgcatgacttttttt 3660 tctaacacaa tgtgtgaaac ctttaataat gatcttgtta agtttattattaagattttg 3720 gtgcggcttt tgtgctacat cgttctctat tgccatgcac caaatatgctgacaactatg 3780 tgtctgggaa ctcttcttgt tttggacatt acaacttgtg aaatcttgtctgccaacacc 3840 aaagcactct ttcaggcatt ggtcgatggt gatgtgaaga gtcttgtctggaaaattgct 3900 gaaaacatgc agtttgccca atccaaagat gaacaagcag aggaaatggcggcaaccttc 3960 aactttgctt ctgatatggt taattttgtg ccaatggaac agatgagacaagaaggctgg 4020 agagaattta atgatgtttc tatgtccttc cggcatgtag aatggtggctgaccatgttt 4080 aaaaaagtgt ataatggtct gaaaagtatt tttgcaccta gtattgaacagaaggctgtt 4140 gattggatag atcgcaatca agaatatatt gccgatgttt tggaccatgcttccaacatc 4200 attataaaaa tgaaggaccc aaaagaacag cggaaagcat taaccattagtgaatacttt 4260 gaagttttga agcaattaaa gccaattgtg tctctttgca tgaaggttgctccctccact 4320 aagttttcct ctcaagtgtt tagaatttat tctgaaatga tgaaggttaatgttagagtg 4380 cctgcaaata ctgacttgac cagattggaa cccattggca tttgggtttctagtgagcca 4440 ggacagggta aatcattttt tacacacatg ttgagcacct gccttttaaaatcctgcaat 4500 ttagagggaa tttataccaa ccccactggg tcagaattta tggatggttatattggacag 4560 gacatccata ttatagatga tgcagggcaa aacagggagg aaaaagatttagccttgttg 4620 tgccagtgta tttcctctgt gccttttacc gtcccaatgg cagatttgacagagaagggc 4680 actttttaca caagtaaaat tgtgattgct accaccaata aatttgattttacatcaatg 4740 gttttgacag atccagcagc tcttgaaagg aggttcccgt ttcatttgcgcattagagct 4800 gtagccagct actcgcgcaa taataaatta gatgtggccc gctcaatggcagccatggct 4860 gatggctctt gctgggaata ctctacagat ggtggtaggg cttggaagactctgtccatg 4920 gatgaacttg tgaaacagat tacggcagtc tatacacaga gatcagatgcccttatggtt 4980 tggaaaagga agttaaacac cattaggaat gaaatgagtc ctggatcctccaccggtagg 5040 atctttgaac ccttggagga aacactttgt gctctggaac gccgctttggtcaacttgct 5100 gatagcctta aagacaatta ccacaaaaca gctgatgagc tgattgaggctatagaagat 5160 atgatggcac catcacagag cccttttgca tgctttgcag aatcctatcggcccaccatt 5220 aaatacactg ccagtgataa agttaaatcc tgggtcaaaa atcatatgaatagatggaaa 5280 gagtttgtaa tgagaaataa aggctggttt acactttttt ctgtgctttcatcttttctt 5340 tcaattctta ctcttgttta cttgcattat aaaaaggaaa agaaagaggaagagagacaa 5400 gagcgagctt acaaccctca aaccgcaact tttaagaagg ggggtaagcccaagctctca 5460 ttggtgaaaa atacaaattt tgttaatgaa gcaccctata tgcaagatcttgaacactgt 5520 tttgcacaaa cagcctacat ctcatcttca gagacccagg atataatacattgtgctgct 5580 ttgagtgaag acaccatctt ggtttatgga cactcccagt tttattttaaccgctatgaa 5640 gatctgcggt tgcattttaa aggggccatt tttcctatag aaggggggaaaatctctcaa 5700 gttactgtga atgggcagcc catggattta attcttgtga aaatagataaacttccaata 5760 acatttaaaa attatacaaa gtactataca actgaggttg gtaaggaaacactcttaatt 5820 tggaattcag agaaaggcag attggctatg cctgttcaat gtgtagccccggctggtccg 5880 gtggaaacaa tggaaggaac agtcacccac aagacctatt catacaaggtggcatcaaaa 5940 aaaggaatgt gtgggggtct cttggttact agagtgcacg gcacatttaaggttttagga 6000 atgcacattg ctggcaatgg acaagttgca cgagccgcag cagtccactttatatccaat 6060 ggggctgctg gctttatgga tcagggtgtt gttgtggcca aggaaaaattgcagaagccc 6120 atttatttgc catccaagac agccttgaat cctagtccct tgaatggagtagttcctgtg 6180 aaaatggagc cagctgtgct tagtcctcat gataccaggc ttgaagttgccatgcccagt 6240 gttgtgaaaa cagcagcagc caagtataga gttaacattt tcaaccctgactttgagatt 6300 tgggagagag ttgtggatga gctaaagtca aggtttagat ttaaacttgggattcataaa 6360 catgtttctt tccaaaaagc agttcagggt ttttcttctc tttcatctcttgatctttcc 6420 acttctccag gacaaaagta tgttgaaaaa ggcatgaaga agagagatcttttatccact 6480 gaaccatttt ggatacatcc tcaattggaa aatgatgtta aagatatacttggggctgtt 6540 tattctggca aaaaacccca tacatttttt gctgcccatt tgaaagatgaattgcgcaaa 6600 aaagaaaaga ttgcacaagg caagacccgc tgcattgaag cctgctcaattgactatgtg 6660 attgcctata gagttgtaat gtcctctctc tatgaggcaa tctatcaaaccccagctcag 6720 gaattaggct tggcagtggg gatgaatccc tggacagact gggatccaatgattaatgtt 6780 ttgcaaccat ataattatgg tttggattat tcatcttatg atggcagtctttctgagcag 6840 ctgatgaggt atggtgtgga aatacttgcc tattgtcatg aacaaccagaggctgtgatg 6900 attcttcatg aaccagttat aaactcacaa caccttgtga tggatgaaatttggcatgta 6960 aatggaggaa tgccctcagg agccccatgt acaactgtgc taaattcyatatgcaatctg 7020 ctggtttgta catatttggc ttatgagcaa agtttggata ttgaggtgttgcccattgtt 7080 tatggagatg atgtgatttt ttccgtttcc tcccctttgg atgctgaatatctggttcag 7140 agtgcagcca gaaattttgg gatggaagtg acctcatcag ataaatctggtcccccaaga 7200 cttttgaaaa tggatgagat tgaattttta aagaggacaa caaaattttttcctggctcc 7260 acctataagg tgggggcctt gagcctggat accatggaac aacatattatgtggatgaag 7320 aatttggaaa cctttccaga acaacttgtt agctttgaaa atgagttggtgttgcatggg 7380 aaagaaattt atgatgatta taaaagtagg tttaatccta ttttgaatcaatggcgagtg 7440 tgcatgcagg actatgaagt ggccctgcat cgcatgctac gctatgtttttgattaaatt 7500 gatttaattt gattttgatt ttgttagttt tagtttaagt aagttagaattagattattt 7560 taatttagct ttaaagattt tgatttgatt gaatttggcc caccaatc7608 4 60 PRT Ljungan virus 4 Gln Ser Arg Tyr Phe Ala Ala Val Arg CysGly Phe His Ile Gln Val 1 5 10 15 Gln Leu Asn Val Asn Ile Gly Ser AlaGly Cys Leu Ile Ala Ala Tyr 20 25 30 Met Pro Lys Thr Ala His Asp His MetAsn Thr Tyr Thr Phe Gly Ser 35 40 45 Tyr Thr Asn Leu Pro His Val Leu MetAsn Ala Ala 50 55 60 5 60 PRT Ljungan virus 5 Ser Phe Arg Cys Pro AsnPhe Phe Phe Pro Leu Pro Ala Pro Arg Glu 1 5 10 15 Ala Thr Ser Arg SerIle Leu Glu Arg Val Asp Glu Ala Asn Ala Glu 20 25 30 Glu Leu Glu Ala ValLeu Glu Ala Arg Thr Pro Asp Ala Pro Leu Arg 35 40 45 Leu Lys Phe Asn ProGlu Asp Pro Leu Lys Gln Leu 50 55 60 6 60 PRT Ljungan virus 6 Pro MetGlu Gln Met Arg Gln Glu Gly Trp Arg Glu Phe Asn Asp Val 1 5 10 15 SerMet Ser Phe Arg His Val Glu Trp Trp Leu Thr Met Phe Lys Lys 20 25 30 ValTyr Asn Val Leu Lys Ser Ile Phe Ala Pro Ser Ile Glu Gln Lys 35 40 45 AlaVal Asp Trp Ile Asp Arg Asn Gln Glu Tyr Ile 50 55 60 7 60 PRT Ljunganvirus 7 Thr Gly Ser Glu Phe Met Asp Gly Tyr Ile Gly Gln Asp Ile His Ile1 5 10 15 Ile Asp Asp Ala Gly Gln Asn Arg Glu Glu Lys Asp Leu Ala LeuLeu 20 25 30 Cys Gln Cys Ile Ser Ser Val Pro Phe Thr Val Pro Met Ala AspLeu 35 40 45 Thr Glu Lys Gly Thr Phe Tyr Thr Ser Lys Ile Val 50 55 60 810 PRT Homo sapiens 8 Ala Leu Gln Cys Gly Arg His Val Asp Val 1 5 10 9 9PRT Homo sapiens 9 Ala Ala Val Leu Pro Gln Thr Ala His 1 5 10 12 PRTHomo sapiens 10 Ala Val Ala Glu Glu Val Asn Ala Ile Leu Lys Ala 1 5 1011 4 PRT Homo sapiens 11 Ile Phe Ser Pro 1 12 9 PRT Homo sapiens 12 GluArg Gly Phe Phe Tyr Thr Pro Lys 1 5

1. A method for obtaining an animal model for human diabetes, comprisingobtaining a mammal and determining whether the mammal is infected with aLjungan virus.
 2. The method according to claim 1, wherein the methodadditionally comprises determining whether the mammal has high bloodglucose levels that can be reduced by insulin and signs of ketoacidosis.3. The method according to claim 1, wherein the method additionallycomprises testing for the presence of autoantibodies to at least one ofGAD65, IA-2 and insulin.
 4. The method according to claim 1, wherein themethod additionally comprises modulating the immune system of the mammalto facilitate the development of diabetes.
 5. The method according toclaim 4, wherein the immune system of the mammal is affected bysubjecting the mammal to stress for at least about 2 months.
 6. Themethod according to claim 5, wherein the mammal is subjected to stressby keeping it in a cage.
 7. The method according to claim 1, wherein themammal is a rodent.
 8. The method according to claim 1, wherein themammal is a bank vole.
 9. The method according to claim 8, wherein thebank vole is Clethrionomys glareolus.
 10. The method according to claim8, wherein the bank vole is obtained from the wild or is the progeny ofa bank vole obtained from the wild.
 11. The method according to claim10, wherein the bank vole is obtained from Denmark, Sweden or Finland.12. A method for using a mammal having high blood glucose levels thatcan be reduced by insulin and signs of ketoacidosis as a model ofdiabetes, wherein the mammal was obtained by the method according to anyone of claims 1 to
 10. 13. (Canceled).
 14. The method according to claim12, wherein the mammal is used to screen for compounds having an effecton symptoms of diabetes.
 15. The method according to claim 14, whereinthe mammal is used to screen for compounds which prevent or reduce thesymptoms of diabetes.
 16. A method for using compounds identified by themethod according to claim 15 in the preparation of a composition fortreating and/or preventing diabetes.
 17. A method for treating a mammalcomprising the step of infecting the mammal with a Ljungan virus and themammal developing diabetes.
 18. The method according to claim 17,additionally comprising suppressing the immune system of the mammal. 19.The method according to claim 18, wherein the immune system issuppressed by subjecting the mammal to stress subsequent to infectionwith the infectious agent.
 20. The method according to any one of claims17 to 19, wherein the mammal is a cat, dog or bank vole.
 21. The methodaccording to any one of claims 17 to 19, wherein the mammal is a bankvole.
 22. A method of using a mammal infected with a Ljungan virus as amodel of diabetes.
 23. A method of using a mammal obtained by the methodaccording to any one of claims 17 to 19 as a model of diabetes.
 24. Themethod according to claim 22, wherein the mammal is used to screen forcompounds having an effect on symptoms of diabetes.
 25. The methodaccording to claim 24, wherein the mammal is used to screen forcompounds which prevent or reduce the symptoms of diabetes.
 26. A methodfor using compounds identified by the method according to claim 25 inthe preparation of a composition for treating and/or preventingdiabetes.
 27. An assay for determining an individual's susceptibility todeveloping diabetes comprising the steps of analyzing a sample from theindividual in order to determine if the individual is infected with aLjungan virus, wherein infection with a Ljungan virus indicates agreater susceptibility to developing diabetes.
 28. The assay accordingto claim 27, wherein the sample is analyzed in order to determine thepresence of a Ljungan virus.
 29. A method of treating an individual whohas developed diabetes or is susceptible to developing diabetescomprising administering an effective amount of a compound whichprevents or reduces the effects of a Ljungan virus, which causes, atleast in part, diabetes.
 30. A method of vaccinating an individualagainst a Ljungan virus infection, thereby preventing, at least in part,the individual developing diabetes.
 31. An isolated nucleic acidmolecule comprising a nucleotide sequence selected from the groupconsisting of: (a) the nucleotide sequence as shown in FIG. 7; (b) thenucleotide sequence as shown in FIG. 8; (c) the nucleotide sequence asshown in FIG. 9; and (d) a fragment of a nucleotide sequence of (a)-(c).32. A polypeptide encoded by the nucleic acid molecule according toclaim
 31. 33. An antibody that specifically binds the polypeptideaccording to claim
 32. 34. The antibody according to claim 33 which ismononclonal.
 35. A vaccine comprising a component selected from: (a) apolypeptide according to claim 32 and (b) an antibody according to claim33.
 36. A method for treating or preventing Ljungan virus infection anddiabetes comprising administering a composition selected from: (a) apolypeptide according to claim 32; (b) an antibody according to claim33; and (c) a vaccine according to claim 35.